MPs Martin Vickers (Cleethorpes) and Edward Leigh (Gainsborough) have joined a growing Tory rebellion which wants to force a House of Commons vote on a European referendum.
They are among more than 50 mostly Conservative MPs who "respectfully regret" that legislation paving the way for a referendum on the UK's membership of the EU was not in last week's Queen's Speech.
Although the Prime Minister has pledged to hold a ballot by the end of 2017 after renegotiating the UK's relations with the EU, the rebels want a legislative commitment sooner rather than later.
Mr Vickers said although he believed David Cameron's stance was "perfectly sensible", the Government had to recognise the "debate had moved on".
He added: "I'm a long-standing Eurosceptic and I will vote no when we have the referendum. Personally I'd have it now – a simple piece of legislation to say there will be a referendum could be passed fairly quickly.
"I would either have it to coincide with next year's European Parliament elections or on the same day as the Scottish independence referendum in September 2014. I've yet to be convinced it can't be held before the next general election in 2015."
And although Grimsby MP Austin Mitchell is absent from Parliament because of illness, he said he "would vote for the Tory amendment" were he able to.
Mr Mitchell, who has joined the "Labour for a Referendum" group, said: "I'm in favour of leaving, and the group exists to demonstrate there are others in the Labour Party also in favour of leaving.
"Europe's in a mess and it's not going to get out of it. I don't see there's anything to be gained now by staying in – there are lots of disadvantages like overpriced food courtesy of the Common Agricultural Policy, the Common Fisheries Policy, and so on."
The Commons amendment was published after two senior Cabinet ministers admitted they would vote "no" if there a referendum on Europe was held this year.
Speaking from Washington DC, the Prime Minister said the existing EU set-up was "unacceptable" but refused to say whether he agreed with Education Secretary Michael Gove and Defence Secretary Phillip Hammond.
"There's not going to be a referendum tomorrow," he said, "there is going to be a referendum before the end of 2017."
But David Cameron will still be in the United States when up to 100 Conservatives are expected to back the amendment on Wednesday.
Although it is unlikely to be passed due to opposition from Labour and the Liberal Democrats, Downing Street confirmed yesterday that Conservative ministers would be able to abstain.
