Labour is to vote against legislation paving the way for a referendum on reforming the voting system.
The shadow cabinet decided to oppose the Government's Bill because it also includes provisions for equalising the size of constituencies.
The move sets the stage for a major test of the coalition, with Labour MPs lining up alongside rebel Tories in a bid to derail the proposals.
The commitment to a referendum on switching to Alternative Vote was a key concession obtained by Liberal Democrat leader Nick Clegg as part of his deal with David Cameron.
But more than 40 Conservative MPs made clear they are against holding the poll on May 5, 2011 as scheduled, because it is the same day as local elections.
If the referendum does not make it through Parliament, disaffection among Lib Dem rank-and-file over the coalition agreement is likely to grow - especially as the party's support appears to have slumped since it was struck.
In an amendment to the Parliamentary Voting System and Constituencies Bill tabled on Tuesday night, the Labour leadership makes clear it still believes in principle that there should be a referendum on AV.
But it insists that the legislation should not be given a second reading "because it combines that objective with entirely unrelated provisions designed to gerrymander constituencies by imposing a top-down, hasty and undemocratic review of boundaries".
Some senior Labour ministers originally suggested that the party's MPs should merely abstain from the vote on the Bill.
However, long-term backers of AV, such as shadow communities secretary John Denham, have now signed up to the tougher approach.
