Rome - Serena Williams withdrew from the Italian Open on Tuesday with a recurring left knee injury.
"Serena Williams has withdrawn with a new knee pain," organisers said.
The 23-time Grand Slam champion's withdrawal comes a day after she beat Sweden's Rebecca Peterson in straight sets to set up a second round meeting with her older sister Venus.
She had been returning after more than two months out with a knee problem, in the final warm-up clay-court tournament before the French Open.
Four-time Rome winner Serena, 37, had eased through 6-4, 6-2 her first round match against Swedish qualifier Petersen.
Serena had also played Petersen in the second round at the Miami Open in March before withdrawing with a knee injury.
Her withdrawal sends Venus Williams, the 1999 winner, through to a third round meeting with either Johanna Konta of Britain or American seventh seed Sloane Stephens.