Alleine belonged to a family that had originally settled in Suffolk. As early as 1430 some of the descendants of Alan, lord of Buckenhall settled in the neighborhood of Calne and Devizes. These were the immediate ancestors of "worthy Mr Tobie Alleine of Devizes", father of Joseph, who was the fourth of a large family, born at Devizes early in 1634. 1645 is marked in the title-page of a quaint old tractate, by an eye-witness, as the year of his setting forth in the Christian race. His elder brother Edward had been a clergyman, but in this year died; and Joseph entreated his father that he might be educated to succeed his brother in the ministry.
In April 1649 he entered Lincoln College, Oxford, and on November 3, 1651, he became scholar of Corpus Christi College. On July 6, 1653, he took the degree of B.D., and became a tutor and chaplain of Corpus Christi, preferring this to a fellowship. In 1654 he had offers of high preferment in the state, which he declined; but in 1655 George Newton, of the great church of St Mary Magdalene, Taunton, sought him for assistant and Alleine accepted the invitation. Almost coincident with his ordination as associate pastor came his marriage with Theodosia Alleine, daughter of Richard Alleine. Friendships among "gentle and simple" of the former, with Lady Farewell, granddaughter of the protector Somerset bear witness to the attraction of Alleine's private life.
His public life was a model of pastoral devotion, yet he found time to continue his studies, one monument of which was his Theologia Philosophica (a lost MS.), a learned attempt to harmonize revelation and nature, which was admired by Richard Baxter. Alleine was no mere scholar or divine, but a man who associated on equal terms with the founders of the Royal Society. These scientific studies were, however, kept in subordination to his proper work. He was surprisingly influential for so young a man, and this was thanks to his earnestness and forcefulness.
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