Serena Williams is out of the French Open after a shock 6-3 7-5 defeat by 21-year-old Elena Rybakina.
In an absorbing match, the 23-time Grand Slam winner was outmuscled by the Kazakh, who will now seek to take advantage of an open women's draw.
The American tried to lift her level but instead became the latest top-10 seed to depart the women's singles.
Her next chance to equal Margaret Court's record of 24 Grand Slam singles titles comes at Wimbledon from 28 June.
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Serena Williams has pulled out of this week's Miami Open after having oral surgery.
The 23-time Grand Slam singles champion was knocked out of the Australian Open semi-finals by Naomi Osaka in January.
"I am disappointed to withdraw," said 39-year-old Williams, who has won the Miami Open eight times.
"Miami is a special tournament for me because it's my home and I am sad I won't be able to see the incredible fans this year."
The men's draw has already been depleted by the withdrawals of world number one Novak Djokovic, Roger Federer, Rafael Nadal, and world number four Dominic Thiem.
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Serena Williams overcame a sluggish start to beat Anastasia Potapova and reach the Australian Open fourth round.
Tenth seed Williams, bidding for a record-equaling 24th Grand Slam title, came through 7-6 (7-5) 6-2 against the 101st-ranked Russian.
However, the American had trailed 5-3 in an error-strewn first set, and she had to save two set points before dialing back in to win.
Naomi Osaka also advanced after a cagey match against Ons Jabeur of Tunisia.
Third seed Osaka and her opponent struggled for rhythm but it was the Japanese who prevailed 6-3 6-2.
Osaka will face two-time Grand Slam champion Garbine Muguruza next, while Williams plays seventh seed Aryna Sabalenka for a place in the quarter-finals.
She will face a tough test against big-hitting Belarusian Sabalenka, who beat American Ann Li 6-3 6-1
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Serena Williams breezed into the Australian Open third round but Petra Kvitova and Bianca Andreescu were the biggest casualties on the third day.
American Williams, who has dropped just five games in her two matches so far, beat Serbia's Nina Stojanovic 6-3 6-0.
Czech ninth seed Kvitova, who reached the final in 2019, was stunned 6-4 1-6 6-1 by Sorana Cirstea of Romania.
Andreescu, playing in her first major since winning the 2019 US Open, lost 6-3 6-3 to tricky Taiwanese Hsieh Su-wei.
Last year's finalist Garbine Muguruza continued her good form on the hottest day of the tournament so far with a 6-3 6-1 win over Russian Liudmila Samsonova.
Belarusian seventh seed Aryna Sabalenka also advanced, beating Daria Kasatkina of Russia 7-6 (7-5) 6-3.
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