Veteran Wales hooker Ken Owens is out of the 2023 Rugby World Cup after failing to recover from a back injury.
The Scarlets front rower captained Wales in this year's Six Nations, but will not go to France this autumn.
Owens, capped 91 times, has not retired from Test rugby, though at 36 years old, his Wales future looks doubtful.
Scarlets back-row Josh Macleod and Cardiff prop Will Davies-King have also been released from Warren Gatland's training squad due to injury.
Ospreys hooker Sam Parry, 31, has been added to the squad in place of Owens.
Gatland said it was "disappointing" to lose the three players, but said they all could be available later in the year for a potential call-up, should Wales suffer injuries.
"We gave Ken as much time as possible but his back has not recovered," he said.
"He might need an operation so he's not going to be available for the World Cup squad.
"He's been a great servant to Welsh rugby, but he wanted to reiterate he has not retired from rugby and he's hoping if he recovers from that and gets himself right on the pitch, potentially he may be available if we pick up injuries during the World Cup."
However, Owens' misfortune may now spell the end of his 13-year international career, which began as an injury call-up during the 2011 World Cup under Gatland.
A British and Irish Lion in 2017 and 2021, Owens has been first choice for Wales for the past seven years.
In recent weeks he has seen long-time Wales team-mates Justin Tipuric, Alun Wyn Jones and Rhys Webb announce their international retirements.
Wales have also seen Cory Hill withdraw as a result of a club offer in Japan, while Rhys Carre was released after failing to hit performance targets.
"Ken is gutted, but he's an incredibly positive person," said Gatland.
"He actually considers himself lucky for getting an extra 18 months that he didn't think he would get after the last back injury.
"He understands that at his age, his body doesn't recover as quickly now, but we've got some strong competition in that position."
Gatland will take his squad to the Alpine resort of Fiesch in Switzerland from 3-17 July.
While Macleod (shoulder) and Davies-King (foot) have been ruled out, Taulupe Faletau will miss the first week due to a calf injury, while Alex Cuthbert and Owen Williams will not travel for "personal reasons".
Gatland said: "We've done a lot of good preparation already and we're heading to Fiesch where we are looking to get the benefits of sleeping at altitude and will kick our training up another gear."
Gatland will then further trim his squad to 38 before a second training camp to Turkey later in July.
Wales captaincy
Gatland has lost more than 400 caps of experience - and four former captains - thanks to the recent retirements, leaving a question mark over who will lead Wales to France in September.
Owens became Wales' oldest Six Nations captain this year and was at the forefront of discussions when Wales' players threatened to go on strike ahead of the game against England in a contractual row with the Welsh Rugby Union.
Gatland has mooted the possibility of appointing joint captains, as pool rivals Australia have done with Michael Hooper and James Slipper.
But he is also open to the idea of doing something similar to 2011, when he made Sam Warburton the youngest captain in World Cup history, aged 23.
"You look at the squad and I don't think there are any guaranteed starting positions [so] co-captaincy is a possibility," said Gatland.
"We did that with Ellis Jenkins and Cory Hill in 2018 and it worked well by sharing the responsibility.
"But we absolutely would consider a young captain [like 2011]. We have time for that with the two camps and seeing more of the rugby."