Former UKIP AM Mark Reckless has left the Conservative group in the assembly, he has announced.
He said he has left the party group because the UK government "has broken its promise to deliver Brexit". Mr Reckless had not been a member of the party.
It comes as speculation mounts a Brexit Party assembly group could be formed.
Tory group leader Paul Davies thanked him "for his valuable contribution" over the last two years.
Mr Davies said: "I appreciate there are strong views on Brexit but our commitment is clear - Wales and the rest of the United Kingdom must leave the European Union."
A spokesman for the South Wales East independent AM, who was declining interviews on Tuesday, said he had left the Tory group on "good terms".
Nigel Farage has formed the Brexit Party to fight the EU elections, which are taking place on 23 May.
Analysis by BBC Wales political editor Felicity Evans
The main thing Mr Reckless' move does is intensify speculation that AMs who've defected from UKIP over the last few years will announce they want to form a new Brexit Party group in the assembly.
Bear in mind that Nigel Farage is visiting Wales again on Wednesday - to Merthyr Tydfil - and Mark Reckless makes this announcement on the eve of that visit.
You don't have to be a genius to work out that two and two makes four.
No one is willing to confirm any of this. But while there's no confirmation, there's no denial either.
And it would be the kind of dramatic flourish that Mr Farage would relish.
One source has suggested the AMs are ironing out who is doing what, and what kind of policies they're going to pursue.
If the group isn't announced, it might be because that has not been sorted out.
There has been no confirmation from any of the individuals - Caroline Jones, Mark Reckless, David Rowlands and Mandy Jones - that are thought could be involved in a new group.
Caroline Jones is known to have signed up to the Brexit Party as a supporter and was at a Newport rally earlier in May, as was Mr Reckless.
One source said Mr Reckless' resignation was a step towards a new group, but did not mean it was a "done deal".
Mr Rowlands, if he was to defect, would leave UKIP with two AMs, not enough to form a party group and would mean it would have to lose its staff.
A UKIP group source said on Monday they had no concerns about Mr Rowlands, however, while a Brexit Party spokesman said a newspaper report of discussions over a new group was a "great work of speculation".
It is the third time Mr Reckless has defected from a political group.
Mr Reckless joined the Welsh Conservative group in 2017 after leaving UKIP, but did not rejoin the party he had served as an MP.
'At face value'
UKIP AM Neil Hamilton said David Rowlands told him and UKIP leader Gerard Batten that he would not be defecting.
Mr Hamilton: "He told me and he told Gerard Batten that he's not going to defect.
"I take his word at face value."
Of the reports and speculation of a Brexit group in the assembly, Mr Hamilton said: "Given the history of the selfishness of those who were elected in 2016 as UKIP AMs to go off and do their own thing, out of frustrated ambition or whatever reason, of course I'm concerned when people betrayed the trust of the party that gave them the opportunity to become AMs and also the people who voted for them in the election.
"I don't think that's a very honest basis to form a new party."
What is a Welsh Assembly group?
AMs generally are members of their party group - the organisation in the assembly which reflects which party they are a member of.
It means that they can nominate people to act as chair-people of committees, have greater speaking rights in the chamber and get extra money to employ staff.
In the assembly a group needs at least three AMs to form.
There are currently four assembly groups - Labour, the Conservatives, Plaid Cymru and UKIP.