In about 1580 or 1581 he was deeply impressed by a sermon, whereupon he retired to the country, and was led by study and meditation to the strictest form of Puritanism. Subsequently, in what manner is not known, he came into intimate relations with John Greenwood, the Separatist leader, whose views he adopted without reserve. Though not strictly resident in London at this time, he was associated with “the brethren of the Separation” there, in whose secret meetings his natural earnestness and eloquence made him conspicuous.
Greenwood having been imprisoned in the Clink, Barrowe came from the country to visit him, and on the 19th of November 1586 was detained by the gaoler and brought before Archbishop Whitgift. He insisted on the illegality of this arrest, refused either to take the ex officio oath or to give bail for future appearance, and was committed to the Gatebouse. After nearly six months detention and several irregular examinations before the high commissioners, he and Greenwood were formally indicted (May 1587) for recusancy under an act originally directed against Papists. They were ordered to find heavy bail for comformity, and to remain in the Fleet Prison until it was forthcoming. Barrowe continued a prisoner for the remainder of his life, nearly six years, sometimes in close confinement, sometimes having “the liberty of the prison.” He was subjected to several more examinations, once before the privy council at Whitehall on the 18th of March 1588, as a result of petition to the queen. On these occasions he vigorously maintained the principle of separatism, denouncing the prescribed ritual of the Church as “a false worship,” and the bisbops as oppressors and persecutors.
During his imprisonments he was engaged in written controversy with Robert Browne (down to 1588), who had yielded a partial submission to the established order, and whom he therefore accounted a renegade. He also wrote several vigorous treatises in defence of separatism and congregational independency, the most important being:—