An amendment expressing regret that the UK government had not included an EU referendum Bill in the Queen's Speech won the support of 130 MPs.
Up to 100 Conservative MPs were expected to vote for an amendment to the UK government's legislative plans, just a week after they were first put before parliament.
The rebels are angry that the legislative programme for the year ahead in the British Queen's Speech did not include proposals for a referendum on Britain's EU membership which has been promised later by British Prime Minister David Cameron.
The party turmoil has fuelled talk of the UK quitting the EU and echoes deep rifts which dogged the Tory party under Margaret Thatcher and John Major.
While the vote is non-binding, a big mutiny would be an embarrassment for Mr Cameron less than two years before the next general election and would boost eurosceptics pushing him to take a harder line on Europe.
Mr Cameron who will miss the showdown because he is in the US played down the row, saying he was "extremely relaxed" about what is a free vote for Conservative MPs, except ministers.
"I keep reading about the number of rebels. I hope someone will define for me how you can rebel on a free vote," he said.
The Labour Party, which has a 10-point opinion poll lead, claimed the revolt showed Mr Cameron was "panicked" over Europe and had lost control of his party.
"We have a prime minister who is not just indecisive, not just weak, but fast becoming a laughing stock," Labour deputy leader Harriet Harman told parliament.