John Knox, in his pamphlet, illustrates the then widely held belief that women were incapable of ruling a country. The mere thought of a woman sitting on the throne in her own right was a break from tradition, as no woman until Mary I had ever been crowned Queen of England in her own right; from Knox's own theological point of belief, women were not capable of being both Head of State and Head of the Church of England.
John Knox, a fervent Protestant Reformer, wrote his pamphlet primarily with Mary of Guise, Queen Dowager of Scotland and regent to her daughter, Mary, Queen of Scots, in mind. Both women were Catholics.
He invoked the Bible as authority for his opinion as "God, by the order of his creation, has [deprived] woman of authority and dominion" and pointed to the historical tradition to back his argument up. Unfortunately for Knox, Elizabeth, a Protestant herself, unsurprisingly disagreed with his views and her opposition to Knox severely hampered his influence with the Protestant movement in England after her succession.