Top seed Amelie Mauresmo crashed out of the Hastings Direct International with a 6-4 6-4 loss to Vera Douchevina.
The defeat was a big blow to the French star's preparations for Wimbledon, where she is seeded three.
Mauresmo, who was warned by the umpire after angrily hitting a ball over the stands, saved three match points before surrendering with a loose return.
"There is nothing much to say. It was a pretty bad day. I should just forget it," said the 25-year-old.
"I didn't quite put my game together - I served and volleyed throughout the match, but on the baseline points I wasn't feeling comfortable at all."
Meanwhile, defending champion Svetlana Kuznetsova survived to reach the quarter-finals, but only after outlasting fellow Russian Anna Chakvetadze 1-6 6-4 7-6 (7-5).
Last year's French Open champion Anastasia Myskina was also taken to three sets before beating Venezuelan Maria Vento-Kabchi 6-4 5-7 6-0.
Mashona Washington benefited from replacing injured Alicia Molik to oust Virginie Razzano 7-6 (7-4) 6-4.