Ed Miliband is facing growing pressure after the leader of the UK's second biggest union called for voters to be offered a referendum on the European Union.
Paul Kenny, the general secretary of the GMB, said the public should get a say on EU membership.
His comments follow warnings by Len McCluskey, the leader of the UK's largest union, Unite, who told The Herald earlier this month that Mr Miliband's refusal to offer a vote could cost Labour next year's general election.
The calls intensify pressure on the Labour leader as his party holds its annual conference in Manchester.
Mr Kenny said: "I think people should have the argument about Europe. I think Labour should offer a referendum on Europe. I have always thought that they would."
He warned that the issue could "hurt" Labour at the general election.
Mr Kenny said that the European Union of which the UK was currently a member had changed considerably in recent years - and that voters should get a say on the future.
It was time to have a discussion about "what the public's views on the European Union are," he added.
Mr McCluskey warned that Labour could lose the next general election if it did not offer voters an EU referendum.
He predicted that the party's political opponents, including the eurosceptic UKIP, would paint Labour as unwilling to trust voters.
In an interview with The Herald he said: "He is going to be portrayed as somebody who is afraid of asking the British people their views, and we think that is tactically dangerous for him."
Asked if the tactic could lose him the election, he said: "In a tight election, it could do, that is exactly right. That is our view. Which is why we would prefer to take a position of saying 'let's call a referendum'.
Mr Miliband has said that Labour will not hold an EU membership vote unless there are proposals to transfer greater powers from London to Brussels.
The move was designed to draw a line under the issue following arguments from some within Labour that the party should meet Mr Cameron's referendum pledge - or risk being punished in May's poll.
Othes warned that offering a vote could tie Labour's hands unnecessarily after next May, and leave it fighting another lengthy and difficult referendum battle just months after the Scottish independence vote.
Mr Miliband's stance appears to have the backing of business leaders.
Earlier this year Sir Michael Rake, the chairman of the CBI, told the Prime Minister that his decision to offer an In/Out referendum was causing "uncertainty" for business.
But others within the Labour movement are pressuring for Mr Miliband to perform a U-turn.
John Mills, who gave £1.5m to the party last year, is also putting money behind a lobbying campaign, Labour for a Referendum.
While many Labour MPs still believe UKIP party still poses the greatest threat to the Conservatives, there is increasing concern about the party's growing popularity in some Labour-held seats.
Mr Cameron has previously said that he would campaign for the UK to stay in the EU.
He has also pledged to renegotiate the UK's relationship with Europe before the 2017 vote.
But reports suggest that the Conservative leader could change his stance - and announce that he will back a Yes vote only if the UK gets a good enough deal from Brussels at his party conference next week.
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