England-based trio Josh Adams, Tomas Francis and Luke Charteris have been released from Wales' summer tour squad.
Wales' contest with the Springboks in Washington DC sits outside World Rugby's designated global window - which begins on the weekend of June 9 and runs until June 24. Ashton Hewitt, Rhodri Jones and Aaron Wainwright have been called up as replacements.
Charteris (Bath), Adams (Worcester Warriors) and Francis (Exeter Chiefs) were initially named in Wales' 31-man squad for the mid-year Tests.
In a further setback, Cardiff Blues back-row forward Josh Navidi has been ruled out of the tour due to a dislocated shoulder suffered during last Friday's European Challenge Cup final victory over Gloucester in Bilbao.
"The policy of the Premiership Rugby clubs is to comply with World Rugby Regulation 9, and only release players for Test matches inside the official Test window", said a Premiership Rugby spokesperson.
A shoulder injury is hampering Josh Navidi
All three players will take no part in Wales' games against South Africa and Argentina and have been immediately replaced in Warren Gatland's touring party.
"We are really disappointed that Josh, Tomas and Luke have to miss out on the opportunity to tour with Wales and play global rugby this summer", Wales head coach Warren Gatland said.
"It was a great opportunity for them to play for their country, press their claim in an important year and it is a shame supporters will not get to see them in the red of Wales".
"The situation is very clear for players playing in England going forward and heading into a RWC year".
A statement from the Welsh Rugby Union confirmed that George North and Ross Moriarty, both of whom are leaving Premiership clubs to join Welsh regions this summer, will still tour.