Dwayne Peel is out of the Barbarians' side to face Wales on 4 June in Cardiff in a row over availability.
Sale's Peel has been named in the Baa-baas squad, but Premiership Rugby say no players from England's top 12 clubs are allowed to do so.
A statement read: "Premiership Rugby has no agreement with the Barbarians and no players will be released to play for the Barbarians [on 4 June]."
Sale confirmed Peel is unavailable to the Barbarians.
"Dwayne Peel will categorically not play for the Barbarians. It is as simple as that," said Sale chief executive Mick Hogan.
"Dwayne has been told and understands the situation.
"Premiership Rugby has negotiated with Scotland and Italy for players to be released from 1 July as long as insurance is in place.
"As far as I am aware Wales have not responded to the offer."
Toulon-bound rugby league convert Willie Mason is due to make his rugby union debut for the Barbarians during their two-match tour.
That could pit him against future team-mate Gavin Henson, who is in the Wales squad and retains hopes of remaining at Toulon next season.
World Cup hopeful Peel had been named in a squad including Scarlets prop Iestyn Thomas and Cardiff Blues flanker Martyn Williams for the clash at the Millennium Stadium.
Wales were not permitted to pick British and Irish Lions scrum-half Peel as English clubs are only obliged to release players for World Cup preparation on 5 August, 2011.
At the time of Peel's selection, Barbarians commercial director Mike Burton told BBC Wales the Baa-baas have an arrangement with the English clubs.
The situation prompted Wales coach Warren Gatland to say: "We were disappointed that we weren't allowed to pick him [Peel] ourselves."
Scarlets prop Thomas was left out of Gatland's Wales squad along with Cardiff Blues flanker Martyn Williams, who had already been named by the Baa-Baas.
Williams will be joined in the Baa-Baas squad by Cardiff Blues team-mate lock Paul Tito - now Welsh-qualified and rumoured to be on the provisional squad list for the World Cup - while Tito's fellow New Zealander, Munster wing Doug Howlett, is also included.
Another former All Black, prop Carl Hayman, is in a squad which boasts France and Toulouse fly-half Frederic Michalack, Italy hooker and former captain Leonardo Ghiraldini and Samoa lock Iosefa Tekori.
Other Barbarians already confirmed for the clash at Cardiff's Millennium Stadium are Italy captain Sergio Parisse, Australia great George Smith and Toulon's South African captain Joe van Niekerk, France centre Mathieu Bastareaud, plus Toulon's England three-quarters Paul Sackey and Iain Balshaw, of Biarritz.
Although Williams and Thomas will be playing against Wales rather than for them, Gatland hinted that the door is still open for World Cup selection.
Wales have three Tests in August - two against England and one with Argentina - before they depart for September's World Cup in New Zealand.
"At 34 Iestyn is not too old to prove he still has what it takes to play at international level and we will be keeping a close eye on him," said Gatland.
"The Barbarians are assembling an impressive squad, they have a long list of quality international players now with the likes of Howlett, Hayman, Michalack and Sergio Parisse in their number.
"We are looking forward to an exciting, free-flowing Test match and we want to put the pressure on and play with tempo and intensity.
"The conditions at the moment will lend themselves to that kind of game and we should have similar weather and hard grounds in New Zealand for the World Cup, so it will give us a good indication of how the players are going to fare."
Gatland has spoken of his interest in seeing Cardiff Blues team-mates Williams and Sam Warburton, who will captain Wales, go head-to-head in this match.
"You definitely get something different when you see players go head-to-head in this way in a proper Test match," added Gatland.
"It's something that you only normally see in derby games but on the international stage it should have an extra edge to it as well.
"With Martyn and Sam both at the same region we haven't had a chance to see them play against each other before and this is definitely a chance for Dwayne to show us what he can do."
Wales squad v Barbarians (Millennium Stadium, Cardiff; 4 June 1430 BST):
Forwards: P James (Ospreys), J Yapp (Cardiff Blues), R Bevington (Ospreys), S Andrews (Cardiff Blues), H Bennett (Ospreys), R Hibbard (Ospreys), L Burns (Newport Gwent Dragons), A-W Jones (Ospreys), R Jones (Ospreys), L Charteris (Newport Gwent Dragons), L Reed (Scarlets), D Lydiate (Newport Gwent Dragons), T Faletau (Newport Gwent Dragons), J Turnbull (Scarlets), S Warburton (Cardiff Blues, capt).
Backs: M Stoddart (Scarlets), G North (Scarlets), A Brew (Newport Gwent Dragons), S Williams (Ospreys), J Davies (Scarlets), S Williams (Scarlets), G Henson (Toulon), S Jones (Scarlets), R Priestland (Scarlets), M Phillips (Ospreys), T Knoyle (Scarlets).
Forwards: S Bruno (Toulon), Q Geldenhuys (Aironi), L Ghiraldini (Benetton Treviso), C Hayman (Toulon), D Kubriashvili (Toulon), E Lund (Biarritz), S Parisse (Stade Francais), S Perugini (Aironi), G Smith (Toulon), I Tekori (Castres Olympique), I Thomas (Scarlets), P Tito (Cardiff Blues), J van Niekerk (Toulon), M Williams (Cardiff Blues).
Backs: B Baby (Clermont Auvergne), M Bastareaud (Stade Francais), D Howlett (Munster), B James (Clermont Auvergne), N Jeanjean (Brive), W Mason (Toulon), F Michalak (Toulouse), R Pienaar (Ulster), S Rabini (La Rochelle), P Sackey (Toulon), S Tillous-Borde (Castres Olympique), T Visser (Edinburgh).