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 Barlow Christ Church and perpetual vice-chancellor, was the ruling power at Oxford during the Protectorate. Among other distinguished associates of Barlow may be mentioned Sanderson, then regius professor of divinity (1642-8), and Robert Boyle, who made Oxford his chief residence (1654-68), whose 'esteem and friendship' he 'gained in the highest degree,' being 'consulted by him in cases of conscience' ('s Life of Boyle, p. 113). Barlow's 'prodigious reading and proportionable memory' rendered him one of the chief authorities of the university on points of controversial divinity and cases of casuistry. He was regarded as 'a great master of the whole controversy between the protestants and the papists,' being the uncompromising opponent of the latter, whose salvation he could only allow on the plea of 'invincible ignorance' (, Genuine Remains, pp. 190-205, 224-31, ed. 1693). He was a decided Calvinist, strongly opposed to the Arminian tenets of Jeremy Taylor and Bull and their school. During this period he was one of the chief champions of what were then considered orthodox views at Oxford, uniting, together with Dr. Tully, a much higher Calvinist than himself, in 'keeping the university from being poisoned with Pelagianism, Socinianism, popery, &c.' (, Athen. Oxon. iii. 1058). Kippis says of him that he was 'an universal lover and favourer of learned men of what country or denomination soever.' Thus we find him 'offering an assisting hand' and showing 'publick favours' to Anthony à Wood, afterwards his ill-natured maligner (, Life, xxiii, lix); patronising the learned German, Anthony Horneck, and appointing him to the chaplaincy of Queen's soon after his entrance at that college in 1663 ('s Life of Horneck, p. 4); helping Fuller in the compilation of his 'Church History,' particularly with regard to the university of Oxford (, Ch. Hist. ii. 293, ed. Brewer); and even 'receiving' at the Bodleian 'with great humanity' the celebrated chaplain and confessor of Henrietta Maria, Davenport, otherwise a Sancta Clara, when visiting Oxford 'in his troubled obscurity' (, Athen. Oxon. iii. 1223). Barlow was by constitution what was contemptuously called a 'trimmer.' Naturally timid, his casuistical training provided him on each occasion with arguments for compliance which always leant to the side of his own self-interest. The freedom with which he regarded some important tenets of the Anglican church is shown by the somewhat depreciating tone in which he spoke of infant baptism in a letter written to Tombes, the anabaptist divine, a letter which, to his honour, he is said to have refused to withdraw when, after the Restoration, it affected his position at the university and damaged his prospect of preferment in the church (, Life of Boyle, p. 299).

On the surrender of Oxford to Fairfax in 1646, Barlow accommodated himself to his changed circumstances without any apparent difficulty. Two years later, when the university was purged of malignants, Barlow was one of the fortunate few who escaped ejection. We may safely set aside Anthony à Wood's spiteful story that he secured the favour of Colonel Kelsey, the deputy-governor of the garrison, by making presents to his wife, and accept the statement of Walker (Sufferings of the Clergy, pt. ii. p. 132) that the retention of his fellowship was due to Selden and his former pupil Owen, then all-powerful in the university, by whom Barlow's learning and intellectual power were justly appreciated. It is certainly surprising, considering his caution against committing himself, except on the winning side, to find him contributing anonymously to the flood of scurrilous tracts issuing from the press on the parliamentary visitation of Oxford in 1648 a pamphlet entitled 'Pegasus, or the Flying Horse from Oxford, bringing the Proceedings of the Visitors and other Bedlamites,' in which, with a heavy lumbering attempt at wit, he endeavoured to hold up the proceedings of the visitors to ridicule. In spite of this indiscretion Barlow retained his fellowship all through the Protectorate, rising from one dignity to another, and finally becoming provost of his college in 1657. He occupied the rooms over the old gateway of the college, now pulled down, which tradition pointed out as those once tenanted by Henry V. On the death of John Rouse, Barlow, then in his forty-sixth year, was elected to the librarianship of the Bodleian on 6 April 1642, a post which he held until he succeeded to the Lady Margaret professorship in 1660, being 'a library in himself and the keeper of another,' 'than whom,' writes Dr. Bliss, 'no person was more conversant in the books and literary history of his period' (, Athen. Oxon. iii. 64). Barlow proved a careful guardian of the literary treasures committed to his charge, opposing 'both on statute and on principle the lax habit of lending books, which had been the cause of serious losses.' He is, however, charged with carelessness in keeping the register of new acquisitions to the library (, Annals of the Bodl. Lib. pp. 79, 84, 100).

On the death of Dr. Langbaine in 1657 Barlow became head of his college. The next year, 1658, we find Robert Boyle