He was acquainted with the Greek text of the New Testament and with Latin commentators, and his antitrinitarian sentiments were the result of his own scriptural studies.
He still continued to preach, publishing a defence of ‘a private man's preaching.’ Early in 1650, he became public preacher to the garrison at Chester, in succession to Samuel Eaton.
The biographer of John Murcot, writing in 1657, speaks of Knowles as having been ‘a formidable and blazing comet at Chester,’ where ‘in public sermons, private conferences, and by a manuscript’ he ‘denied Jesus Christ to be the Most High God.’ A short paper of arguments for the deity of Christ, sent by Eaton to Chester from Dukinfield, was published by Knowles in 1650, with his own reply.
The imprimatur of Porter's pamphlet, entitled ‘A Serious Exercitation,’ is dated 26 December 1650, and by that time Knowles was ‘late preacher at Chester.’ He appears to have returned to Gloucester, for on 19 November 1650, the mayor of that city was directed by the council of state to examine witnesses on oath respecting Knowles's preaching against the divinity of Christ.
On 23 June and again on 7 July he petitioned (writing also to George Monck, 1st Duke of Albemarle) for liberty to go out on bail, as the plague was then raging in London.