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Ambrose Barlow was born Edward Barlow in 1585 at Barlow Hall in Chorlton-cum-Hardy, near Manchester, in Lancashire, England. He was a son of Sir Alexander Barlow, head of a recusant gentry family that remained Catholic through the Elizabethan settlement and the penal laws of the late Tudor and early Stuart period. Recusant households such as the Barlows faced fines, exclusion from public office, and the risk of imprisonment. Edward was baptized in the parish church as English law required, but he was raised in the Catholic faith. The family's attachment to that faith was reflected in the vocations of several of his brothers; the most notable was Rudesind Barlow, who became a leading superior of the English Benedictines in exile on the Continent.

Because Catholic education and ordination were forbidden in England, Barlow went abroad in the early seventeenth century. He studied at the English College at Douai in the Spanish Netherlands and at the Royal College of St Alban in Valladolid, Spain, colleges founded to train clergy for the English mission. He then entered the Benedictine community of St Gregory at Douai, took Ambrose as his name in religion, and was professed as a monk of the Order of Saint Benedict. He was ordained priest in 1617 and almost at once accepted assignment to the English mission. Under a statute of 1585 it was high treason for a priest ordained overseas to enter the kingdom, and those convicted were liable to be hanged, drawn, and quartered.

Barlow worked for some twenty-four years chiefly in south Lancashire, a region where recusancy remained comparatively strong among both gentry and yeomen. He moved between Catholic halls and farmhouses, offering the sacraments, instructing the young, and assisting the poor and the sick. Contemporary accounts describe him as gentle, patient, and unpretentious. He was arrested and imprisoned on several occasions, yet each time he returned to the same countryside. In later years he suffered from dropsy, which added to the physical hardship of missionary life. On Easter Sunday in 1641, while celebrating Mass at Morleys Hall in Astley, he was seized by a local Protestant clergyman and an armed party and taken to Lancaster Castle.

At the Lancaster assizes he was indicted as a seminary priest. He did not deny his priesthood and refused offers of mercy that required him to conform to the established church. On 10 September 1641 he was hanged, drawn, and quartered at Lancaster. His death fell in a year of mounting political and religious tension, only months before the outbreak of the English Civil War. Catholics at once regarded him as a martyr. Pope Pius XI beatified him on 15 December 1929. Pope Paul VI canonized him on 25 October 1970 as one of the Forty Martyrs of England and Wales, a group chosen to represent Catholics put to death in England and Wales between the 1530s and the late seventeenth century. His liturgical memorial is kept on 10 September. Relics associated with him have long been preserved in Lancashire, and he is honoured especially in the Manchester area where he was born and where he spent his missionary years.

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