Olympic silver medallist Marketa Vondrousova withdrew from the Paris Games due to a hand injury, the Czech 25-year-old said, with Poland's Hubert Hurkacz also withdrawing.
Vondrousova reached the women's final in Tokyo three years ago as an unseeded player, losing to Swiss No. 9 seed Belinda Bencic. The Swiss, who gave birth to a daughter in April, will not be in Paris to defend her title.
"I am very sorry, but due to health reasons I will not be participating in this year's Olympic Games in Paris," Vondrousova posted on Instagram.
"I hoped until the last moment that I could go at least in doubles, but problems with my hand won't allow me on the court."
Katerina Siniakova, who won gold at the last Olympics in doubles and is a nine-time Grand Slam champion in women's doubles, will replace Vondrousova in the singles and Linda Noskova will play doubles with Karolina Muchova.
Vondrousova won her first Grand Slam at Wimbledon last year, the first unseeded woman to win the singles title, but became the first defending Wimbledon women's champion to exit in the first round for 30 years.
Hurkacz was forced to retire from his Wimbledon second-round match earlier in July when he dove for a volley but landed badly and injured his knee, and has not recovered in time to compete in Paris.
"My rehabilitation is going very well, and I am making continuous progress. However, my team and I have decided that I am unable to compete in the Olympic Games in Paris," Hurkacz said in a video posted on Instagram.
"This was a very, very difficult decision because I have always dreamed of representing Poland at the Olympics, being able to win a medal for the national team, but unfortunately, my health does not allow it this year."
Other notable absences from tennis at the Games include world No. 3 Aryna Sabalenka, Ons Jabeur, Emma Raducanu, Ben Shelton and Tokyo men's bronze medal winner Pablo Carreno Busta of Spain.