World No.3 and Australian Open champion Angelique Kerber was dumped out 6-4 6-2 by Barbora Strycova at the Mutua Madrid Open.
Joining her on the sidelines was top seed Agnieszka Radwanska, beaten in three sets by the ever dangerous Dominika Cibulkova.
Cibulkova has now beaten the world No.2 in three of their last five meetings.
Kerber, after suffering a post-Australia slump, had rebounded with semifinal finishes in Miami and Charleston before defending her title in Stuttgart.
Yet she was no match for the crafty world No.33; Strycova will next face Madison Keys in the second round after Keys rallied past fellow American Alison Riske 1-6 6-3 6-4.
With top-ranked Serena Williams pulling out of Madrid due to illness, the tournament is now devoid of the world’s top three women.
Seventh-seeded Italian Roberta Vinci lost 6-4 6-2 to Danka Kovinic, while last year’s finalist, No.9 seed Svetlana Kuznetsova, fell in three sets to the in-form Laura Siegemund.
Vinci, Kuznetsova and Radwanska were all in the top quarter of the draw; that section is now entirely devoid of seeds after 15th seed Sara Errani crashed out in straight sets to fellow Italian Camila Giorgi.
Home favourite Garbine Muguruza, the third-seeded Spaniard, had little trouble advancing past Anna Karolina Schmiedlova 6-2 7-5, breaking her serve six times.
Another Spaniard, eighth-seeded Carla Suarez Navarro, fought back against Timea Babos to win 3-6 6-2 6-3.
Karolina Pliskova, seeded 13th brushed off Lourdes Dominguez Lino 6-2 6-1.
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