Opponents of Jeremy Corbyn accused Unite of an "attempted stitch-up" after the union offered a vote in the Labour leadership contest for as little as £2-a-month.
The union said that affiliates, who would have full voting rights, could still sign up.
Overnight Labour looked to have barred from voting more than 100,000 people, many of whom joined the party in the last few weeks.
Only those who have been members since January 12 will be eligible.
Individuals will also have a two-day window next week to become 'registered supporters', for a £25 fee.
The sum is a significant hike on last year, when £3 guaranteed a vote.
But Unite, which backs current leader Jeremy Corbyn, said those who wanted to vote could join for as little as £2-a-month for students and the unemployed until August 8.
Those earning more than the minimum wage would pay just over £14-a-month.
Sources also said that members would have to quit after just a short time to make any savings.
But a Labour source denounced the move as an "attempted Unite stitch-up".
Labour's rules were changed last year in response to allegations of vote-rigging in Falkirk linked to Unite.
"You could not make this up", a source said.
The row erupted as a second 'unity' candidate emerged against Mr Corbyn.
Welsh MP Owen Smith said that he would be a "radical and credible" leader who could return Labour to Downing Street as he confidently predicted he would win.
Labour MPs attempts to oust Mr Corbyn suffered a major setback yesterday when he automatically became a candidate in the race.
His opponents had hoped he would be forced to find 51 nominations from MPs and MEPs.
Some argued that was unlikely following the collapse in his support among MPs in recent weeks.
More than 50 members of the shadow cabinet have walked out in protest at what they say was his lacklustre leadership in the EU referendum.
Some accuse him of running a half-hearted campaign because of his own qualms about EU membership.
He famously used an appearance on a television chat show to give the EU “seven out of 10” just weeks before the vote.
But Mr Corbyn’s supporters insist he was in touch with much of Labour’s voters, millions of whom rejected its message to stay in the EU.
Meanwhile, a member of the party's ruling body the NEC accused Mr Corbyn of endorsing bullying, threats and intimidation of his opponents by voting against a secret ballot that would decide whether ot not he would automatically be a candidate.
But Mr Corbyn defiantly insisted he was enjoying “every moment” of being Labour leader.
Pontypridd MP Mr Smith told BBC Radio 4's Today programme that it was right that Mr Corbyn took part in the race.
In comments that raised eyebrows among some of this colleagues, he also accused some on the right of the party of acting in a way which could "split" Labour.
He said: "I will stand in this election and I will do the decent thing and fight Jeremy Corbyn on the issues, just as he will do with me, and at the end of that I will stand behind whoever the leader is.
"But I hope and I expect it will be me."
But he said that he oppose attempts to hold Tony Blair in contempt over his decision to go to war in Iraq.
At the weekend Mr Corbyn said that he would “probably” support such moves.
Mr Smith was not an MP during the 2003 Iraq War vote.
The other candidate, Angela Eagle also launched a call for the campaign to be free from abuse.
Earlier this week she a brick was thrown through her constituency office.
Shadow chancellor John McDonnell said that Mr Corbyn had also suffered death threats.
He rejected suggestions that calling Mr Corbyn’s opponents "plotting and conniving" and describing them as “f****** useless" was abuse, insisting it has been a joke.
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