Olympic men's doubles champions Bob and Mike Bryan have pulled out of Rio 2016 to protect their "family's health".
The American duo's announcement comes after fellow tennis stars Milos Raonic, Simona Halep and Tomas Berdych withdrew from the Games over the Zika virus.
In a statement, Bob Bryan said he and his brother made the decision after "countless hours of deliberation".
"Though we'd love to compete again, as husbands and fathers our family's health is now our top priority."
The Bryan brothers, 38, made no direct reference to Zika in their statement.
Women's world number five Halep and men's world number seven Raonic were the first tennis players to make themselves unavailable because of Zika, which is linked to brain deformities in newborn babies.
More than 20 male golfers have also withdrawn from Rio 2016, with many concerned about the mosquito-borne virus.
The Florida-based Bryan brothers won Olympic gold at the London 2012 Games - beating France's Jo-Wilfried Tsonga and Michael Llodra in the final - and own 112 titles together, including 16 Grand Slams.
"Representing Team USA is one of our proudest tennis moments to date and winning gold in 2012 will always remain the pinnacle of our career," their statement added.
"The Olympics mean so much to us for so many reasons and the memories we have wearing the Stars and Stripes will burn brightly forever."