Williams, who won at Istanbul two years ago, was ousted in straight sets by Aravane Rezai, 6-4, 6-4. Rezai had won just five previous matches this year.
Second-seeded Elena Dementieva and No. 3 Patty Schnyder advanced to the quarterfinals with straight-set victories Wednesday. Dementieva stopped Jelena Kostanic Tosic 6-3, 6-3 and Schnyder was a 6-4, 6-1 winner over Vania King.
Also in second-round play, fifth-seeded Alona Bondarenko needed a third-set tiebreaker to get past Anastasija Sevastova 6-4, 3-6, 7-6 (7-5); No. 7 Agnieszka Radwanska sailed past Romina Oprandi 6-1, 6-0; Meghann Shaughnessy whipped Ekaterina Afinogenova 6-1, 6-3; and Catalina Castano overcame Ekaterina Dzehalevich 4-6, 7-5, 6-1.
Quarterfinals play was set for Thursday for the French Open tune up.