Bath will not release Welsh internationals Gavin Henson and Paul James for the 'Probables' v 'Possibles' trial match on May 30.
A club spokesman confirmed that the club will follow Premier Rugby Limited regulations, where players do not have to be released for non-Test matches not covered by the IRB.
As a result the club are sticking with the PRL rules, meaning both players will not be released despite their seasons finishing a week earlier with the Challenge Cup Final in Cardiff.
"As it stands, we are unable to release the players for the game on the 30th, due to the test window regulations that all 12 Aviva Premiership Clubs agreed to via Premiership Rugby," said a club spokesman.
The participation of other English-based players including Owen Williams, George North, Ryan Jones and Rhys Gill is now also in doubt, although Williams, Gill or North could be playing in the Premiership Final.
North is the only player to have his release for all Wales matches written into his contract.
The news would appear to rule out Henson's chances of playing on the tour of South Africa, given that the trial match was viewed as an opportunity for him to impress Warren Gatland. He has not played for Wales since 2011.