More than 100 MPs expressed their regret tonight at the Government's decision not to include an EU referendum Bill in its new legislative programme.
The 130 MPs backed an amendment to the Queen's Speech, tabled by Tory Eurosceptics John Baron and Peter Bone, in a House of Commons vote.
Mr Baron's amendment was signed by 92 MPs, mostly Conservative Eurosceptics. But, despite the support, it was defeated by 277 votes to 130, majority 147.
Prime Minister David Cameron had said he was relaxed about tonight's vote and offered Tory backbenchers and unpaid Government aides a free vote.
Ministers were told they were allowed to abstain in the vote on Mr Baron's amendment, which expressed regret that an EU referendum Bill was not included in the Government's planned legislation.
And in a further effort to win over Eurosceptics, the Conservative Party yesterday published a draft EU Referendum Bill paving the way for an in/out vote before the end of 2017.
As the Liberal Democrats oppose the measure, the legislation will have to be brought forward as a Private Member's Bill. The ballot is tomorrow morning and it is expected that the Bill will be picked up by a well-placed Tory Eurosceptic.
There were gasps in the Commons as the number of MPs to vote for Mr Baron's amendment was read out.
There had been reports during the day that the number of MPs prepared to vote for the amendment had dropped off after the promise to introduce a draft EU referendum Bill following tomorrow's Private Members' Ballot.
Mr Baron had pledged to stick with his amendment despite being unable to get 100 MPs to sign it. But in the end far more than 100 voted in favour of the amendment.
Despite Mr Cameron offering the concession of a free vote to backbenchers and Government aides, tonight's vote will probably be seen as the most significant since October 2011 when 111 MPs, around 80 of them Conservative, revolted over Europe in a bid to exert pressure for an in/out referendum.
Earlier, in the Commons debate on the Queen's Speech, Mr Baron, the MP for Basildon and Billericay, said if the Government pushed a Bill in its own time, it would ensure a majority of MPs would be needed to stop it.
He said Mr Cameron's preferred option of a Private Member's Bill could be easily defeated.
Mr Baron said: "If we were to pass this amendment tonight, we as a Parliament would be opening the possibility to introducing legislation that would stand a far better chance of succeeding.
"It would take a majority to defeat that legislation rather than a determined minority on a Private Member's Bill."
But Chancellor George Osborne told MPs there was a good chance that if the Bill was adopted by an MP it would safely pass through Parliament.
He said: "We now have draft legislation for an in/out referendum on the EU, we have now done it in good time for this session's Private Members' Ballot. It is now open to any MPs who do well in that ballot to adopt the draft Bill we published yesterday, take it forward as the basis for legislation.
"And, as the Prime Minister said yesterday, we will do everything we can to make it the law."
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