Top-ranked Serena Williams suffered a shock 6-4 6-4 loss to Slovakia's world number 78 Jana Cepelova in the second round of the Family Circle Cup on Tuesday.
The world number one and defending champion lost the first five games and never recovered.
"Obviously things didn't go great out there, but she was a great counterpuncher today and hit a lot of balls back," Williams told reporters.
"I really knew nothing about her game going into the match, so she played a really good match against me today. And it just wasn't my day," added Williams.
The 20-year-old Cepelova had never beaten a top 15 player but held her nerve to pull off the biggest win of her career.
Williams, a 17-time grand slam champion, won the Sony Open on Saturday and had been on a roll coming to Charleston, South Carolina where she was a three-time champion.
Her sister Venus also had problems in her opening round match, losing seven consecutive games at one point, but recovered to beat Barbora Zahlavova 6-3 0-6 7-5.
Venus, who won the event in 2004, said she had been under the weather recently.
"I've actually been pretty sick lately, so it was a little rough out there today," she said. "I just kept fighting till the end."
In other early-round action, sixth seed Eugenie Bouchard breezed past Alla Kudryavtseva 6-2 6-0, while Australian seventh seed Samantha Stosur overcame a stiff challenge from Yaroslava Shvedova in a 6-3 5-7 6-1 triumph.
Eighth seed Sorana Cirstea was upset by Teliana Pereira in a 3-6 6-3 7-6(0) defeat.