Naomi Osaka suffered a second-round exit at the Madrid Open on Sunday as the Japanese second seed lost 6-4, 3-6, 6-1 to Karolina Muchova.
Osaka, the reigning US and Australian Open champion, has seven career hard-court titles, but has never managed to lift a clay-court trophy.
Playing her first clay-court event since the 2019 French Open, Osaka beat compatriot Misaki Doi in the first round, but she came unstuck against an opponent making her top 20 debut this week.
"I was ready to play with all I had today, and to use all the weapons I could," Muchova said. "I'm definitely happy it worked out."
Muchova beat three top 20 players, including world number one Ashleigh Barty, en route to the Australian Open semi-finals earlier this year and she can now add world number two Osaka to her growing list of scalps.
"I was trying to move her as much as possible," Muchova said. "That was the tactic. It sounds easy but she's playing really fast so it's not that easy on the court.
"In the second set especially, she started to be very aggressive. I got back to what I did in the first set in the third set, and again, tried to move her left, right, dropshots, but still in a fast way."
Muchova used a single break of serve to claim the opening set before Osaka, a quarter-finalist at Madrid two years ago, fell 3-1 behind in the second but rallied to win the next five games and force a decider.
Muchova regrouped and broke Osaka early in the third set, and then again for a 5-1 lead, before sealing victory in just under two hours.
The Czech will meet Greek 16th seed Maria Sakkari, who brushed aside Estonia's Anett Kontaveit 6-3, 6-1.
Fifth seed Aryna Sabalenka also made it through to the last 16 with a 6-3, 6-3 win over Daria Kasatkina.