Second-seeded Angelique Kerber lost 6-4, 6-2 to Barbora Strycova in the first round of the Madrid Open on Sunday.
Heading into their clay-court opener, this year's Australian Open champion hadn't dropped a single set to Strycova in four prior meetings.
But the third-ranked Kerber committed three double faults and was broken by Strycova four times to fall in a little over an hour.
The 33rd-ranked Strycova will face Madison Keys in the second round after she rallied past fellow American Alison Riske 1-6, 6-3, 6-4.
Home favorite Garbine Muguruza, the third-seeded Spaniard, had little trouble advancing past Anna Karolina Schmiedlova 6-2, 7-5, breaking her serve six times. Their match was plagued by poor serving by both players, who combined for 12 double faults.
Also, seventh-seeded Roberta Vinci lost 6-4, 6-2 to Danka Kovinic, while eighth-seeded Carla Suarez fought back 3-6, 6-2, 6-3 against Timea Babos, and 13th-seeded Karolina Pliskova brushed off Lourdes Dominguez Lino 6-2, 6-1.