World number one and top seed Angelique Kerber suffered an unexpected defeat to Estonian qualifier Anett Kontaveit in the second round of the Internazionali BNL d'Italia in Rome.
The German, who earlier this week replaced Serena Williams at the top of the WTA rankings, went down 6-4 6-0 against 21-year-old Kontaveit, the world number 68.
Kerber, who was handed a first-round bye, produced 22 unforced errors in her defeat, which took less than an hour.
"Everybody knows I am not a clay-court specialist," Kerber told wtatennis.com.
"I think I need one good match to also get my confidence back and then to see that it works, actually.
"Because I am working hard, I'm trying to practice really hard and trying to improve the movement, especially on clay, because it's not 100 per cent.
"I am not feeling so well on this surface."
Fourth seed Dominika Cibulkova of Slovakia was also a surprise casualty, going down 1-6 6-1 6-3 to Russia's Ekaterina Makarova.
There was no such drama for number two seed Karolina Pliskova, however, as the Czech player beat American Lauren Davis 6-1 6-1 to take her place in the last 16.
Third seed Garbine Muguruza Blanco of Spain is also through after beating Latvia's Jelena Ostapenko 2-6 6-2 6-1.
Venus Williams saw off Lesia Tsurenko of Ukraine in straight sets, 6-4 6-3, and the American will next face Johanna Konta, the British world number six.
However, 14th seed Barbora Zahlavova Strycova is out after the Czech lost 6-4 6-2 against Timea Bacsinszky. The Swiss will next take on Pliskova for a place in the quarter-finals.
Earlier on Wednesday, number six seed Simona Halep of Romania beat German Laura Siegemund 6-4 6-4 and Elina Svitolina, the eighth seed from Ukraine, defeated France's Alize Cornet 6-4 7-6 (13/11).
Holland's 15th seed Kiki Bertens is also through after beating Catherine Cartan Bellis of the United States 6-4 6-0, while 12th-seeded Russian Anastasia Pavlyuchenkova beat Latvia's Anastasija Sevastova 7-6 (7/4) 6-2.