Top seed Ana Ivanovic crashed out of the Italian Open in her first game on Wednesday, losing 6-4, 5-7, 6-2 to Bulgarian qualifier Tsvetana Pironkova.
Former world number one Venus Williams beat Australian Samantha Stosur 6-4, 6-1 in a game postponed from Tuesday because of rain to join Pironkova in the third round.
Serbia's Ivanovic made an untidy start with too many groundstrokes finding the net or going long and she soon went down a break, but she broke back in the ninth game only to fail to hold immediately after and concede the set.
The world number three twice surrendered a break advantage in the second set and let a string chances to take the Bulgarian's serve again go begging before she finally managed to level things up.
But the unforced errors kept flowing and she was broken three times on the way to losing the decider.
Seventh seed Venus Williams, returning from over a month on the sidelines with an unspecified medical problem, was put to the test immediately against Stosur and lost her serve in the opening game.
She broke back in the eighth game and cruised after that, breaking Stosur again to take the first set before dominating the second.
Fifth seed Serena Williams, who like her sister Venus is a former Italian Open champion, faces Ukrainian Alona Bondarenko later on Wednesday, another match that was bumped over from Tuesday.
Russian world number two Maria Sharapova will start her Rome campaign later against Slovak Dominika Cibulkova.
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