A second government aide today revealed he may join the mass Tory revolt in Monday's Commons vote over Europe.
Aidan Burley, who is parliamentary private secretary to Transport Secretary Justine Greening, said he wants a referendum on whether Britain should stay in the EU.
"I have not decided what I am going to do," he said. "I need to think about it and to reflect local opinion.
"I am of the view that there should be a referendum at some point to resolve this question of Europe but I must also consider whether this is the best time, bearing in mind the importance of inward investment."
David Cameron will meet parliamentary private secretaries, the ministerial aides on the lowest rung of the ladder, on Monday afternoon in a last effort to avert resignations.
The dramatic move came as the Tory whips unleashed a weekend operation to minimise what is expected to be the biggest rebellion yet for the Prime Minister, when MPs divide over holding a referendum.
Five parliamentary private secretaries are said to be considering their positions, with Stewart Jackson the only one so far to say he will definitely vote against the Government.
Mr Cameron is denying claims that he raised the stakes unnecessarily by threatening to sack rebels. Allies say the battle was forced on him.
"The idea that it would have blown over if there had been just a one-line whip is total rubbish and they know it," said a No 10 ally.
"The Prime Minister has responded, not with weakness, but with strength."
More than 60 Tories are backing a rebel motion demanding a referendum. Today an amendment went down from the eurosceptic Tory MP George Eustice, who is seen as friendly with No 10, proposing a White Paper first on what powers might be repatriated from Brussels.
The new motion could provide an escape hatch for MPs nervous of being painted as extremists who want to quit the EU. However, it was attacked by rebel leaders as a Government ploy to divide the rebels.
Mark Pritchard, secretary of the 1922 Committee of backbenchers, said: "The amendment would kick it into the long grass. I cannot support it."
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In an email to MPs, Mr Eustice suggested the amended motion "more closely reflects the views of the majority of the parliamentary party".
Labour splits emerged today. MP Graham Stringer accused leader Ed Miliband of a "mistake" in subjecting the party to a three-line whip, and said it was time people were given a choice.
