Serena Williams suffered a surprise loss to Argentina's Nadia Podoroska on her return to court at the Italian Open on Wednesday.
The veteran American, who was seeded eighth in her first tournament for three months, was beaten 7-6 7-5 in what was the 1,000th match of her illustrious career.
It was Podoroska's first meeting against the 23-time grand slam winner and she came through in one hour and 58 minutes in Rome.
"It's a special win," the 24-year-old told the WTA Tour website. "To play against her, it's an honour for me. I saw her playing when I grew up, so it was a special match for me."
Williams, 39, was pleased to be back in action but felt there was plenty of room for improvement ahead of the upcoming French Open.
"It's tough to have a first match on clay," she said. "It was definitely kind of good to go the distance and to try to be out there, but clearly I can do legions better."
Simona Halep's French Open hopes are in peril after she was forced out by a calf injury.
The Romanian third seed, defending champion in Rome, was leading 6-1 3-3 against fellow former world number one Angelique Kerber when forced to retire.
"Unfortunately an ultrasound has revealed that I have a tear in my left calf," Halep tweeted. "I will get an MRI to understand the injury in more detail, but at the moment we are unsure of recovery time."
Kerber will now face former French Open champion Jelena Ostapenko, who beat qualifier Ajla Tomljanovic 6-2 7-6.
World number two Naomi Osaka is also out after being beaten 7-6 6-2 by American Jessica Pegula.
Pegula survived three set points before taking the first set but then broke twice to take the second.
World number one Ashleigh Barty cruised through 6-4 6-1 against Yaroslava Shvedova, while reigning French Open champion Iga Swiatek beat Madison Keys 7-5 6-1.
