Many members of the higher clergy regarded their relationship with the Church purely in economic terms, and 40% of known illegitimate births (ie those subsequently legitimized) were the product of the 'celibate' clergy's liaisons.

Another spur to the Scottish Reformation was the identification of Protestantism with anti-French feeling. In 1554, Mary of Guise, the French mother of the absent Queen Mary, had become regent, and her habit of appointing Frenchmen to high office was seen as part of an attempt to subordinate Scotland's interests to those of France. There was considerable resentment, and in 1557 a group of nobles banded together to form the Lords of the Congregation, whose dual purpose was to oppose French influence and promote the reformed religion. With English military backing, the Protestant lords succeeded in deposing the French regent in 1560, and, when the Scottish Parliament assembled shortly afterwards, it asserted the primacy of Protestantism by forbidding the Mass and abolishing the authority of the pope. The nobility proceeded to confiscate two-thirds of Church lands, a huge prize that did much to bolster their new beliefs.

Even without the economic incentives, Protestantism was a highly charged political doctrine. Luther had argued that each individual's conscience was capable of discerning God's will. This meant that a hierarchical priesthood, existing to interpret God's will, was unnecessary and that the people themselves might conclude their rulers were breaking God's laws, in which case the monarch should be opposed or even deposed. This point was made very clearly to Queen Mary by the Protestant reformer John Knox at their first meeting in 1561. Subjects, he told her, were not bound to obey an ungodly monarch.

Knox, born in East Lothian, had returned to Scotland in 1559 from exile. He was a follower of the Genevan reformer Calvin, who combined Luther's views on individual conscience with a belief in predestination. Calvinism argued that an omnipotent God must know everything, including the destinies of every human being. Consequently, it was determined before birth who was to be part of the Elect, bound for heavenly glory, and who was not, a doctrine that placed enormous pressure on its adherents to demonstrate by their godly behaviour that they were of the Elect. This was the doctrine that Knox brought back to Scotland and laid out in his Articles of Confession of Faith, better known as the Scot's Confession, which was to form the basis of the reformed faith for over 70 years.

Mary ducked and weaved, trying to avoid an open breach with her Protestant subjects. The fires of popular displeasure were kept well-stoked by Knox, however, who declared 'one Mass was more fearful than if 10,000 enemies were landed in any part of the realm'. At the same time, Mary was engaged in a balancing act between the factions of the Scottish nobility. Her difficulties were exacerbated by her disastrous second marriage to Lord Darnley, a cruel and politically inept character, whose jealousy led to his involvement in the murder of Mary?s favourite, David Rizzio, who was dragged from the queen's supper room at Holyrood and stabbed 56 times. The incident caused the Scottish Protestants more than a little unease, but they were entirely scandalized in 1567, when Darnley himself was murdered and Mary promptly married the Earl of Bothwell, widely believed to be the murderer. This was too much to bear, and the Scots rose in rebellion, driving Mary into exile in England at the age of just 25. The queen's illegitimate half-brother, the Earl of Moray, became regent, and her son, the infant James, was left behind to be raised a Protestant prince. Mary, meanwhile, became perceived as such a threat to the English throne that Queen Elizabeth I had her executed in 1587.

Knox could now concentrate on the organization of the reformed Church, or Kirk, which he envisaged as a body empowered to intervene in the daily lives of the people. Andrew Melville, another leading reformer, wished to push this theocratic vision further. He proposed the abolition of all traces of Episcopacy - the rule of the bishops in the Church - and that the Kirk should adopt a Presbyterian structure, administered by a hierarchy of assemblies, part-elected and part-appointed. At the bottom of the chain, beneath the General Assembly, Synod and Presbytery, would be the Kirk session, responsible for church affairs, the performance of the minister and the morals of the parish. In 1592, the Melvillian party achieved a measure of success when presbyteries and synods were accepted as legal church courts and the office of bishop was suspended.

James VI disliked Presbyterianism because its quasi-democratic structure - particularly the lack of royally appointed bishops - appeared to threaten his authority. He was, however, unable to resist the reformers until, strengthened by his installation as James I of England after Elizabeth's death in 1603, he restored the Scottish bishops in 1610. The argument about the nature of Kirk organization would lead to bloody conflict in the years after James's death.