Top seed Agnieszka Radwanska suffered a first-round exit at the Aegon International after she was beaten 6-4 3-6 7-6 (7/4) by Russian Anastasia Pavlyuchenkova.
Radwanska won the grass-court tournament in 2008, but she has since fallen in the first round four times and not won a match at Eastbourne since she reached the quarter-finals in 2011.
Despite her early exit, Radwanska was not too concerned about her lack of match preparation for Wimbledon. In 2012 after a first-round defeat she went on to reach the Wimbledon final, and last year after an opening-round loss she reached the semi-finals there.
"The first match (on grass) is always tricky," she admitted. "Well, it was a great match before Wimbledon to get used to it. It was a really good match, actually. I just hope I can do the same good results (at Wimbledon) this year."
Defending champion Elena Vesnina of Russia held off a strong challenge to beat China's Shuai Peng 6-3 6-4 and there was another hard-fought victory for 2012 runner-up Angelique Kerber, who beat Alison Riske of the United States 7-6 (8/6) 6-4.
Eighth seed Caroline Wozniacki also made a winning return to the WTA Tour in a high-profile first round encounter with former US Open champion Sam Stosur.
Wozniacki prevailed 7-5 6-4 to set up a second round showdown with American Sloane Stephens, a 6-2 6-4 winner over France's Caroline Garcia.
Eastbourne also features world No 8 Victoria Azarenka as the Belarussian returns from a left foot injury that restricted her to just one match since she surrendered her Australian Open title in the quarter-finals in January.
Drawn to face Italian Camila Giorgi in her opening match on Tuesday, Azarenka said, "I'm very excited. I can't wait to play.
"It's not important for me who's going to be on the other side. I just want to see what I can do, and just see how to raise my level from there.
"I have been tested in practice, but competition is a completely different thing."
Everyone's mind inevitably is on Wimbledon, which begins next week, but second-seeded Petra Kvitova, who reached the Eastbourne final in 2011 before going on to claim the Wimbledon title, is determined to keep her focus on this week.
"Of course, I want to play the best that I can to get used to the grass and everything before Wimbledon, but I'm taking this tournament as a very big one," she said. "It's a very tough draw. Everybody wants to be successful here."