Hantuchova has never been past the last eight at Amelia Island
Croatian qualifier Karolina Sprem enjoyed a surprise win over third seed Daniela Hantuchova at Amelia Island.
Former world number 17, Sprem, ranked 296 following a recent return from a wrist injury, came through 6-3 1-6 6-3.
Former world number one Amelie Mauresmo took a tentative step towards finding her form with a 7-5 6-1 victory over Romania's Sorana Cirstea.
Lindsay Davenport continued her good run by beating fellow American Jill Craybas 6-1 6-4 on the green clay.
"It was a fine match for me to play," said Davenport.
"Overall, I felt I actually hit the ball better than I did in the previous match and did a few things better and now it's just about trying to get more consistent."
Hantuchova was full of praise for Sprem, who injured her wrist at the Italian Open in May and did not return to the tour until two weeks ago - winning two matches in qualifying this week.
"I don't think I did too many things wrong," insisted the Slovakian.
"She was just playing incredibly, going for every shot and almost everything going inside the court. She was just too good."
Mauresmo, who hinted at retirement earlier this year after a poor run of form, has yet to go further than round three at any event in 2008 and has a 13-7 record for the year so far.
Second seed Anna Chakvetadze of Russia beat Japan's Ayumi Morita 6-3 6-1 but conceded: "I didn't really play as well as I wanted to play.
"But I think for the first match in this tournament and the first match on green clay, it was OK. I could do worse."