Williams has lost three matches in a row for only the second time in her career
World number two Serena Williams suffered a surprise defeat by Patty Schnyder at the Italian Open.
After a rain-interrupted day in Rome, the pair did not get onto court until after 11pm and Schnyder eventually sealed a 6-2 2-6 6-1 win at 1am.
Williams had described herself as "the real number one" and "the best in the world" at a news conference on Monday.
Dinara Safina, who replaced Williams at the top of the rankings last month, beat Virginie Razzano 7-6 (7-1) 6-1.
Williams, 27, was playing her first match in almost a month and appeared to be moving with difficulty at times.
They tell us go and play because they think the court is fine but they are not playing on it. They have to have a little bit of respect for us as players
Schnyder had only won three of her previous 11 meetings with Williams but, tellingly, both their encounters on clay, including a victory in Rome two years ago.
Williams has now lost her last three matches - only the second time this has happened in her career.
Safina lost to 10-time Grand Slam champion Williams at the Australian Open in January and has been criticised in some quarters for achieving the top ranking without winning one of the four major titles.
But the Russian, 23, insisted: "It's not luck. Nobody gave it to me - I proved it. I played two Grand Slam finals, and last year I won four titles, so I think I belong to this, because this doesn't come out of the sky."
On Williams, Safina added: "She's older than me, so she has more experience than me, so let's see when I'm her age how many titles I'm going to have, and then we can say."
And the Russian had sympathy for all her fellow competitors regarding the difficult conditions at the Foro Italico.
"I was just so angry because you can get injured," she said. "They tell us go and play because they think the court is fine but they are not playing on it.
"They have to have a little bit of respect for us as players."
Third seed Jelena Jankovic
went through after beating Argentine Gisela Dulko 6-3 7-6 (8-6), despite a four-hour rain break, while fifth seed and French Open champion Ana Ivanovic
beat Francesca Schiavone 6-3 6-4.
"It's difficult when it rains and then you have to stop and focus again and start," said Serbia's Jankovic.
"I had everything under control, and all of a sudden she's on top of me and she has set points to win the second set.
"It was difficult but I managed to somehow stay positive and stay strong and win that second set."
who beat Safina to win the Stuttgart title on Sunday, saw off Daniela Hantuchova 6-3 6-3, and ninth seed Caroline Wozniacki
defeated Bethanie Mattek-Sands 6-1 7-5.