Wimbledon champion Petra Kvitova suffered a 6-3 7-6 (7-2) defeat by American Madison Brengle on her return to the WTA Tour in Stuttgart.
Kvitova, 25, was playing her first WTA match since February following
The Czech did last weekend but was error-strewn in Stuttgart.
Meanwhile, defending champion and top seed Maria Sharapova lost 2-6 7-5 6-1 to Angelique Kerber of Germany.
Both were knocked out of the second round after being given byes in the first round.
Danish fourth seed Caroline Wozniacki, who has brought former French Open champion Arantxa Sanchez-Vicario into her coaching team, beat Czech Lucie Safarova 7-6 (7-5) 6-1.
Third seed Kvitova made 22 unforced errors in the first set as the far steadier Brengle broke serve once but created six break points.
The 25-year-old from Delaware is at a career high of 43 as she enjoys her best season to date, and she broke again at the start of the second to take a grip on the match.
Brengle let a 40-0 lead and four match points slip when she served for the match at 5-4, but she recovered to dominate the tie-break and converted her sixth match point.
"I think that Madison played a solid match," said Kvitova, who made 39 winners and the same number of errors.
"She didn't miss much and it was really difficult for me because when I had the opportunity for winners I missed. The confidence wasn't really high then and I didn't feel physically there as well.
"I was still tired probably from the Fed Cup and on the clay you need to be physically ready for it. So, that was one of the reasons as well.
"I need to be still patient and I'm looking forward to next week, which I'm going to practise on clay."
Sharapova's defeat was her first at the venue after sweeping to the title in 2012, 2013 and 2014 on the back of 13 successive wins.
It also means the 28-year-old French Open champion will lose her world number two ranking to Simona Halep next week.
Kerber, the world number 14, will face Sharapova's Russian compatriot Ekaterina Makarova in the quarter-finals.