Italian Flavia Pennatta inflicted more misery on the Williams family by eliminating elder sister Venus 7-5 6-3, just hours after Serena's third-round defeat to Katarina Srebotnik.
The 26th seeded Pennetta completed victory in fading light and will play Spanish qualifier Carla Suarez Navarro in her first last 16 match at Roland Garros.
Despite a confident start which saw the American eighth seed break in the second game, Pannetta broke back on four occasions to clinch the opening set after 45 minutes.
The Italian continued to take advantage of Williams' unforced errors at the start of the second set.
Pennatta broke early and then went on to clinch the match on Williams' serve with a clinical forehand winner.

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