World number one Iga Swiatek saw her 37-match winning streak ended as France's Alize Cornet caused one of the biggest upsets so far at Wimbledon.
The 21-year-old from Poland was beaten 6-4 6-2 in an inspired performance by 37th-ranked Cornet.
Cornet, 32, coasted to a famous victory by winning the final six games.
Coco Gauff was also knocked out as fellow American Amanda Anisimova fought back from a set down to reach the fourth round for the first time.
Anisimova, seeded 20th, will play France's Harmony Tan, who added Britain's Katie Boulter to her list of scalps at the All England Club.
World number 115 Tan beat seven-time Wimbledon singles champion Serena Williams in round one, eliminated Spain's 32nd seed Sara Sorribes Tormo in the second round and thrashed Boulter 6-1 6-1 on Saturday.
Whoever wins between Anisimova and Tan will face either Spanish fourth seed Paula Badosa or Romania's 2019 champion Simona Halep in the quarter-finals.
Badosa reached the last 32 with a 7-5 7-6 (7-4) win over two-time champion Petra Kvitova, while 16th seed Halep defeated Poland's Magdalena Frech 6-4 6-1.
That half of the draw has opened up following Swiatek's defeat by Cornet, who goes on to face Australia's Ajla Tomljanovic.
Swiatek has become the dominant player on the WTA Tour this year, having won her sixth tournament in a row at the recent French Open.
Her previous defeat came back in February when she lost to Jelena Ostapenko in Dubai.
The streak enabled her to take over from the retired Ashleigh Barty as the world number one, with her recent success giving her a considerable lead at the top of the rankings.
Despite winning the girls' title at the All England Club in 2018, Swiatek admits she does not feel as comfortable on the grass as the clay and hard surfaces.
"I know I didn't play good tennis. I was pretty confused about my tactics," said Swiatek on what wrong against Cornet.
"As a solid player, she used that pretty well. For sure, it wasn't a good performance for me."
The two-time French Open champion did not play a tournament on grass before going to Wimbledon, citing a shoulder injury as she pulled out of Berlin.
Taking a break to recover mentally from her exploits at Roland Garros was important to Swiatek, but her lack of confidence on the grass showed in a performance which lacked fluidity.
"Here, I know how I felt before matches, I know how I felt when I was practising," said Swiatek
"Let's just say that I didn't feel like I'm in a best shape. So I'm kind of aware that this could happen.
"I tried many things to feel better on court, on grass court, but it didn't really work out."