World No 1 and defending champion Serena Williams was handed a shock first-round defeat by Jana Cepelova at the Family Circle Cup in Charleston on Tuesday.
The American top seed was seen off in straight sets as the Slovakian registered a 6-4 6-4 victory in an hour and 25 minutes to reach the second round.
A disappointed Williams, a three-time winner of the tournament, admitted she struggled to find form in her service game.
"I was really struggling with my serve,” she told familycirclecup.com. “I didn't really get any big ones in, so I took the pace off. It wasn't my day. It wasn't meant to be."
Meanwhile, there was swift progress for Eugenie Bouchard who advanced to the third round with a convincing win over Alla Kudryavtseva.
The Canadian took just 58 minutes to overwhelm the Russian qualifier 6-2 6-0.
Samantha Stosur is also safely through following a 6-3 5-7 6-1 victory over Yaroslava Shvedova of Kazakhstan, but eighth seed Sorana Cirstea was dumped out following a 3-6 6-3 7-6 defeat to Brazilian Teliana Pereira.
While Czech Lucie Safarova defeated Virginie Razzano of France 2-6 6-4 6-4 to reach the second round.
In first-round action, 11th seed Venus Williams avoided following her sister in bowing out as she overcame a second-set collapse to beat Barbora Zahlavova Strycova 6-3 0-6 7-5.
Others seeds to advance on day two included Daniela Hantuchova, who beat Shelby Rogers 7-5 7-5 and Elena Vesnina, who was a 6-2 6-1 victor over Anna Schmiedlova.
Andrea Petkovic, the 14th seed, claimed a 6-7 6-4 6-1 over Lesia Tsurenko and Shuai Zhang overcame Hsieh Su-wei 3-6 7-6 7-6.
America’s Vania King beat Israeli Julia Glushko, while Spaniard Lourdes Dominguez Lino overcame compatriot Anabel Medina Garrigues.
Marina Erakovic of New Zealand and China’s Shuai Peng also booked a place in the second round with victories over Nadia Petrova and Caroline Garcia respectively.