Top seed Ana Ivanovic suffered a shock defeat to Tsvetana Pironkova at the Rome International.
Ivanovic was beaten 6-4 5-7 6-2 by the Bulgarian qualifier, who troubled the world number three throughout with her powerful hitting.
The Serbian was able to save five set points from 2-5 and 0-40 down in the opening set, but Pironkova eventually held her nerve to take it at the sixth attempt.
Ivanovic grabbed the decisive break in the 11th game of the second set, but the third was one-way traffic as Pironkova booked her place in the third round with the biggest win of her career.
Her previous best victory came over Venus Williams at the 2006 Australian Open.
"I definitely made a lot of unforced errors off both sides. It felt tough, it was quite hard out there," Ivanovic said.
"I struggled to find my rhythm or find something inside to fight. I struggled to find my intensity today.
"I felt coming into this tournament that I was in good shape, I was working hard and doing well so I'm very disappointed. I really didn't play that well today."
Wimbledon champion Williams had no problems reaching round three in Rome, as she defeated Samantha Stosur 6-4 6-1.
The American seventh seed will meet Vera Zvonereva next after the Russian scraped through 7-5 4-6 7-5 against Iveta Benesova.
Venus' sister Serena, seeded fifth, wasted little time against Ukrainian Alona Bondarenko, hammering the doubles specialist 6-2 6-0.
Second seed Maria Sharapova also progressed in the tournament, seeing off the challenge of Dominika Cibulkova 6-2 3-6 6-4.

Fleming - ATP had vested interest
Davydenko masters Nadal
Davydenko v Nadal
Nadal v Lopez
Davydenko v Djokovic
Djokovic happy with straight sets victory
Nadal v Blake - Nadal
Del Potro wins US Open
The countdown to the ATP World Tour Finals continues on Sky Sports with the ATP Shanghai Masters.
Skysports.com caught up with former Wimbledon champion Pat Cash to discuss all things US Open 2009.
Gerry Williams told Sky Sports News he is delighted that Justine Henin will return to competitive tennis.