What was the main religious difference between the Separatists and Puritans?

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The main difference between the two is that the ordinary Puritans wanted to reform the Church of England from within. Despite their numerous misgivings, they were prepared to remain members of the Church but would endeavor to purify its practices to keep them more in line with their rigid Calvinist standards.

As for the Separatists, they believed that the Church was simply incapable of reform. They didn't regard the Church of England as a true church, at least not as they understood it. In common with the more mainstream Puritans, they argued that the Church had departed from the ideals of the Reformation. However, unlike them, the Separatists felt that the Church had strayed so far from the path of righteousness that it was now completely beyond the pale. As such, they deemed it necessary to break away from the Church of England entirely and establish a completely new church based upon a strict adherence to Scripture.

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