Third-seeded Williams, who reached the 2002 final at Roland Garros, lost to No. 29 Agnes Szavay of Hungary 6-0, 6-4. It was only the 14th time in 662 career matches that Williams lost a set at love.
"I'm used to beating people 6-0," Williams said. "I'm not used to my shot not going in and losing a set 6-0. So it completely was foreign ground for me."
The seven-time Grand Slam champion was playing for the third straight day. She'd needed three-setters to win her first and second rounds and saved a match point against Lucie Safarova on Thursday.
"I had a lot of struggles out there and ultimately you've got to play well," Williams said. "Today it just didn't come together for me."
Maria Sharapova and her injured right shoulder managed to earn yet another three-set win. The unseeded Russian lost the first set but rebounded to beat Yaroslava Shvedova of Kazakhstan 1-6, 6-3, 6-4 and advance to the fourth round.
"I dug a nice pothole for myself there ... so many errors and so many mistakes," Sharapova said. "Then, got rid of those errors and started playing better."
Defending champions Ana Ivanovic and Rafael Nadal had little trouble in their matches, both dominating their opponents. Top-seeded Dinara Safina also won easily in the women's draw.
Nadal defeated former No. 1 Lleyton Hewitt 6-1, 6-3, 6-1 and Ivanovic beat Iveta Benesova of the Czech Republic 6-0, 6-2.
Ivanovic, who lost in the 2007 French Open final before winning last year, has conceded only eight games since being taken to a tie-breaker in her opening match.
"(The) score doesn't indicate how hard I had to work for some points," said Ivanovic, a former No. 1.
first Grand Slam
Safina, the women's No. 1, defeated Anastasia Pavlyuchenkova of Russia 6-2, 6-0.
Safina, trying to win her first Grand Slam title after losing in the final at Roland Garros last year and in the Australian Open final this year, has lost only four games in three rounds.
Novak Djokovic reached the third round in the men's tournament, quickly completing his suspended match against Sergiy Stakhovsky of Ukraine 6-3, 6-4, 6-1.
Fourth-seeded Djokovic won the first two sets on Thursday before darkness stopped play. He broke Stakhovsky to open the third set and had little trouble the rest of the way.