Jordan Spieth will not compete in the Olympic golf tournament, International Golf Federation president Peter Dawson has announced.
His withdrawal means the world's top four golfers will not compete in Brazil, with Jason Day, Dustin Johnson and Rory McIlroy having previously said they would not take part.
Spieth - who won The Masters and US Open in 2015 - confirmed the news as he continued his preparations for The Open, which takes place at Royal Troon.
Golf still has eight of the top 15 players in the world competing at the Olympics, although the loss of the world's top four players could prove to be a major setback as the sport looks to retain its place in the Games in future.
It is assured of being part of the Tokyo Games in 2020, but the International Olympic Committee votes next year to decide whether it stays beyond that.
The USA still have four players in Rio, the most of any country. Spieth's place will be taken by Matt Kuchar, who reached No 15 in the world rankings two weeks ago with a tie for third in the Bridgestone Invitational.
Bubba Watson, Rickie Fowler and Patrick Reed are the other Americans taking part.
Spieth was enthusiastic about the Olympics until about a month ago, when he began to voice concerns over security and Zika.
Johnson withdrew on Friday, with Spieth now following suit just hours ahead of the midnight deadline on Monday in Scotland for those considering pulling out, prompting Dawson to describe the withdrawals as an "overreaction".
So far, South Africa's Lee-Anne Pace is the only female player to pull out.
"We have invested a huge amount of time and effort on player education and they've had no lack of opportunity, I think, to make their own well-informed decisions about what they want to do," he said.
"It's certainly disappointing that we've had so many withdrawals on the men's side, and wonderful that all of the women have been very supportive.
"There is no doubt that the number of withdrawals hasn't shown golf in the best light and we have to accept that. But we do understand why these individual decisions have been taken.
"Personally, I think there's been something of an overreaction to the Zika situation, but that's for individuals to determine. I have no knowledge that people are using Zika as an excuse.
"I think there is a genuine concern about this, not just amongst the players but among their families, their wives and their girlfriends.
"What I'm hoping is that when we come to play in Tokyo in 2020 that the top players do support Olympic golf. I think it's very important that they do.
"It's the biggest grow-the-game opportunity available and I can't think of a better way for players to give back to the game, frankly, than to support Olympic golf."
France's Victor Dubuisson and Korea's KT Kim also withdrew on Monday, but Dubuisson wrote on Twitter that his decision was due to poor form and that he would prefer to give his place to compatriot Julien Quesne.
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