Second seed Elina Svitolina and Coco Gauff were beaten in the second round of the Eastbourne International.
Ukraine's Svitolina, 26, lost 6-4 7-6 (7-3) to French Open quarter-finalist Elena Rybakina of Kazakhstan.
American Gauff, 17, started strongly against lucky loser Anastasija Sevastova, but was defeated 4-6 7-5 6-2 by the 31-year-old Latvian.
It was a setback for Gauff the week before Wimbledon, where she had a fairytale run to the last 16 in 2019.
There was also disappointment for Canadian third seed Bianca Andreescu, who was beaten 6-3 6-3 by Estonia's Anett Kontaveit.
Another leading player to make an early exit was Swiss sixth seed Belinda Bencic, who was beaten 6-4 6-4 by compatriot Viktorija Golubic.
Birmingham Classic finalist Daria Kasatkina continued her great grass form, beating Polish fourth seed Iga Swiatek 4-6 6-0 6-1.
Belarusian top seed and 2018 finalist Aryna Sabalenka did advance, though, beating American Alison Riske 6-1 6-4.
In the men's tournament, British wildcard Liam Broady staged a spirited fightback but lost 6-3 6-4 to Australian second seed Alex de Minaur, while top seed Gael Monfils of France was also knocked out.
There was British success in the men's doubles for Luke Bambridge and Jamie Murray, who beat French fourth seeds Fabrice Martin and Eduoard Roger-Vasselin 6-4 6-4.
Lloyd Glasspool, partnering Finland's Harri Heliovaara, defeated Australians Max Purcell and Luke Saville 6-2 6-3.
Joe Salisbury and his American partner Rajeev Ram, the third seeds, beat Frederik Nielsen and Franko Skugor 7-5 6-3.
Jonny O'Mara and partner Hugo Nys, from Monaco, ended a packed schedule of play, beating British brothers Ken and Neal Skupski 2-6 6-3 10-8.
The Eastbourne International is the last grass-court stop in the UK before Wimbledon starts on Monday, 28 June.