Warren Gatland has expressed his disappointment at three England-based players missing out on Wales' summer tour.
Worcester wing Josh Adams, Exeter prop Tomas Francis and Bath lock Luke Charteris will not be part of the 30-man group.
Wales' June 2 clash against South Africa in Washington D.C. falls outside World Rugby's June Test window, and England-based players will not be made available by Premiership Rugby for the Springboks fixture.
The Welsh Rugby Union announced that Worcester wing Adams, Exeter prop Francis and Bath lock Charteris have been released from the squad as they were not available for the whole tour, which also features appointments with Argentina on June 9 and 16 in South America.
The official Test window next month is from June 9-23.
"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," a Premiership Rugby spokesman told Press Association Sport.
Dragons wing Ashton Hewitt and his fellow uncapped teammate, forward Aaron Wanwright, have been called into the squad, along with Ospreys prop Rhodri Jones.
Wales head coach Gatland said: "We are really disappointed that Josh, Tomas and Luke have to miss out on the opportunity to tour with Wales and play international rugby this summer.
"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 Rugby World Cup year.
"With the players unavailable to link up with the squad until the week of our second Test match, we have had to replace them in the squad."
The WRU said Northampton wing George North and Gloucester back-row forward Ross Moriarty, who are returning to Wales to join the Ospreys and Dragons respectively this summer, will tour.
Gatland had already decided not to select the vast majority of Wales players who toured New Zealand with the British & Irish Lions last summer.
His original squad contained a solitary uncapped player in Blues scrum-half Tomos Williams, but he is now joined by Hewitt and Wainwright.
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.
Reflecting on Navidi's absence, Gatland added: "It is a blow for Josh to pick up the injury and miss out on the summer.
"He has been an important player for us over recent campaigns and has really impressed."