Serena Williams suffered a shock defeat to Italy's Francesca Schiavone in the second round of the Rome Masters in Wednesday's night match.
The American, who had a bye in the first round, crumbled in the face of a partisan crowd and lost 7-6 (7-2) 6-1.
In another surprise, fourth seed Elena Dementieva earlier went down 7-5 6-4 to Argentina's Gisela Dulko.
Meanwhile, defending champion Amelie Mauresmo cantered to a 6-2 6-0 victory over Australia's Samantha Stosur.
"I felt it was close at the beginning, then I put my level up a little higher. 6-2 6-0 is a great way to start the tournament," said Mauresmo.
Elsewhere, Russians Vera Zvonareva and Nadia Petrova had wins over Jelena Jankovic and Mara Santangelo respectively.
Williams provoked whistles from the Rome crowd when failing to run for a drop shot in the opening set tie-break against Schiavone.
And the American continued to struggle in the second set as she succumbed to the world number 26.
"I couldn't get my legs moving," said Williams. "It was really weird. I've never felt like this before.
"I actually had a really good warm-up right before the match and I thought everything would come together, but it didn't."
Asked how she rated the defeat in terms of her career, Williams said: "I think everyone in their career has their worst loss and this is probably it for me - for sure.
"And the last. It won't be happening again."
Schiavone said: "I feel like Popeye beating Brutus."
Williams had not planned to play another tournament before the start of the French Open on 23 May but may rethink her schedule, admitting: "I didn't expect this to happen."