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Although the University Library suffered, as did all other English libraries, terrible destruction and neglect during the Reformation and the years that followed it, some volumes from its earliest years still survived. Thus the library still possess one of the books bequeathed by Loring in 1416 [MS. Dd.7.17], three other books listed in the catalogue of 1424–1440 (including an illuminated Boethius, De consolatione, with Chaucer's translation [MS. Ii.3.21]), fifteen others that had entered the library by 1473, four manuscripts and thirty-five printed books certainly or probably given by Rotherham, and three manuscripts and twelve printed books given by Tunstal, including his Homer (though this volume was alienated from the library at an unknown date and returned to it through the generosity of a benefactor in 1918). Although, therefore, a catalogue of the library drawn up for Cardinal Pole's commissioners in 1557 lists fewer than 200 volumes, all of them gathered together into the east room, the survival of so many is emphatically more remarkable than the loss of the remainder; and it may be added that the wind of change, if it took many books from Cambridge, also brought to it four manuscripts that had belonged to Balliol College, Oxford, including a Thucydides in Latin bequeathed to it by one of the most distinguished early English collectors, William Grey.

There is, however, no evidence that the University Library was deliberately and spectacularly purged of its contents. Rather it would seem that it was eroded meanly and by degrees because its books were thought to be irrelevant to the times and no longer useful; and most illuminating of all is the bleak fact that for forty-five years after Tunstal's benefaction there is no official record of any donation or bequest to the library or of any expenditure on the maintenance of its furniture and fittings. Books were, in fact, safer in private than in public hands. We need not doubt that some books were stolen by men who wished to destroy them because they thought their doctrine dangerous or evil; but it is equally certain that others were taken away—sometimes even officially borrowed—by men who wished to preserve them. Thus Roger Ascham borrowed in 1539–1540 but never returned a manuscript that soon after 1600 found its way into the library of Trinity College, and Sir John Cheke similarly borrowed between 1540 and 1543 three manuscripts that never came back, one of them passing eventually into the possession of Queen Christina of Sweden and so into the library of the Vatican. In the same way we certainly owe the preservation of Crome's surviving manuscript to Andrew Perne, who removed them from the library and kept them privately for thirty years or more until it was once again a place to which he might safely restore them.

Andrew Perne, Master of Peterhouse from 1554 until his death in 1589, was a man who preferred continuity to controversy, and though he excited derision by successfully accommodating his principles to those which it was politic to hold under the successive reigns of Edward VI, Mary, and Elizabeth, he was for more than forty years a central and influential figure in the university, and it is to him that we owe the restoration of the University Library in 1574. He saw that, if such a thing were to be done and the necessary books procured, he must first engage the support of an impressive patron who might give the lead to others. To that end he turned to Matthew Parker, Archbishop of Canterbury and the most eminent and influential bookman in all England, and then, through Parker, to Sir Nicholas Bacon (Lord Keeper of the Great Seal), Robert Horne (Bishop of Winchester), and James Pilkington (Bishop of Durham). These four benefactors agreed to furnish between them, subject by subject, a representative collection of the most recent works of scholarship put out by the great publishing houses of Europe. Thus Horne gave fifty volumes of the Fathers; Pilkington twenty volumes of histories; Bacon ninety-four volumes (in each of which was placed an armorial gift plate, believed to be the earliest English specimen of its kind) of philosophy, grammar, rhetoric, astronomy, geography, music, and mathematics; Parker himself seventy-five volumes of Protestant theology and twenty-five manuscripts, including several of the old English chroniclers and six in Anglo-Saxon. Of his manuscripts the most important and celebrated are perhaps MS. Ii.2.4 (Gregory's Pastoral Care in King Alfred's translation, written ca. 1050–1075, almost certainly at Exeter), MS. Ii.2.11 (the Gospels in West Saxon, of about the same date, presented to Exeter by Bishop Leofric), MS. Ii.4.6 (Aelfric's Homilies, ca. 1050), and MS. Ff.1.27 (a mixed manuscript, part twelfth- and part fourteenth-century, of miscellaneous histories including Gildas and Nennius). All these books, and what remained from the pre-Reformation library, were set up in Rotherham's east room (the south room having been evacuated of books in 1547), all the new books except some of Parker's being chained to their lecterns in the fashion still prevailing from earlier times. A catalogue drawn up in 1583 shows that the volumes numbered about 450 and enables their arrangement to be ascertained with detailed exactitude.

This fair beginning soon stimulated other benefactions. In 1581 the French reformer Theodorus Beza sent to the library from Geneva the fifth-century manuscript of the Gospels and Acts in Greek and Latin which is still its most treasured possession, the Codex Bezae Cantabrigiensis (MS. Nn.2.41)—a manuscript so deviant from the normal that Beza himself thought it "a book to keep rather than to publish" lest its eccentricities should give offence to the faithful. Perne himself gave in 1585 (or bequeathed in 1589) well over a hundred manuscripts, about half of which had once belonged to the library of Norwich Cathedral Priory. Before the end of the century there were also added about 140 books of medicine bequeathed by Thomas Lorkyn, Regius Professor of Physic, in 1594; and eighty-seven volumes, mostly of theology and history, which were duplicates from his own shelves (many of them had once belonged to Thomas Cranmer), were given by John, Lord Lumley, in 1598 in fulfilment of a promise he had made to Perne ten years earlier. Thus, with the library's holdings at last approaching 1,000 volumes, the south room was again taken into use as a library.

Meanwhile, the chaplaincy of the university having been abolished as a relic of popery in 1570, the office of University Librarian with a yearly stipend of £10 was created in 1577. In the beginning it was not an office of any great repute since, a financial crisis almost immediately supervening, the university was compelled to appoint to it such persons (including during 1587–1593, an honest but illiterate tradesman of the town) as might be satisfied with a reduced salary of £3. 6s. 8d.; and when in 1601 the university drew up a memorandum of its chain of command for the guidance of its new chancellor Sir Robert Cecil, it listed its librarian among the "Ministers for the necessary Use of the University" and placed him lower in that section than the auditors, printers, appraisers, and vintners. At the same time, in 1582, the university agreed the first set of regulations governing the conduct of the library and the duties of the librarian. There was to be a triple inventory of the library's contents, of which the vice-chancellor was to hold one copy, the University Chest another, and the librarian a third. All manuscripts and books with coloured pictures, all globes and mathematical instruments and all valuable books of mathematics and history were to be locked up under two keys, of which the vice-chancellor was to hold one and the librarian the other. The librarian himself was to attend in the library during term, except on Sundays and holidays, 8–10 a.m. and 1–3 p.m.; he was to see that all necessary repairs were promptly carried out and that all books were closed and in their right places when he went off duty; he was to give a bond of £200 against the proper performance of his duties and was to replace lost or mutilated books "or else lose his office and pay the triple value". Admission to the library was confined to masters of arts, bachelors of law or physic, and doctors, but only ten persons (excluding sightseers) might use it at one time, and no reader might study the same book for more than an hour if it was wanted by another.

Image: Codex Bezae Cantabrigiensis, MS. Nn.2.41. The ending of St John’s Gospel and the opening of St Luke.