Shadow foreign secretary William Hague has appeared to commit the Tories to a referendum on the new EU treaty even if Parliament has already ratified it.
He said a lack of democratic legitimacy "would not be acceptable to a Conservative government and we would not let matters rest there".
But a close aide to Tory leader David Cameron insisted that there had been "no change" in his party's position.
Europe Minister Jim Murphy said Mr Cameron had "caved in" to rightwingers.
In 2005, Labour, the Tories and the Lib Dems promised a referendum on the now defunct EU constitution.
The Tories say the treaty is largely the same but Labour insists it is not.
Thrown out
During the Commons debate on last week's Queen's Speech, Mr Hague said: "We all promised, all political parties in this House, promised a referendum before the ratification of this treaty."
Mr Cameron has criticised Prime Minister Gordon Brown for refusing a referendum and insists the treaty is almost identical to the Constitution thrown out in 2005 by Dutch and French voters.
But despite pressure from Tory Eurosceptics, he has stopped short of pledging to hold a referendum even if the treaty has been ratified by the time of the general election.
Commenting outside the Commons, Foreign Secretary David Miliband said: "This is proof that Euroscepticism in the Tory Party is back with vengeance.
"A referendum once the treaty has been signed and sealed by all 27 member states would be bad for Britain, leave us isolated in Europe and would mean years of instability and uncertainty."
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