Roberto Bautista Agut was the highest-profile casualty on day two of the Barcelona Open, the fifth seed losing in three sets to Karen Khachanov.
World number 17 Bautista Agut had been expected to progress with a degree of comfort against an unheralded teenage opponent who has rarely featured in the main draws of ATP World Tour events to date.
However, after serving impressively en route to taking the first set, Khachanov overcame the setback of losing a subsequent tie-break to triumph 6-2 6-7 (4-7) 6-3.
The three other seeds in second-round action on Tuesday all progressed, although Alexandr Dolgopolov (11) - Khachanov's next opponent - was forced to save a match point in the second set before edging out Evgeny Donskoy 4-6 7-6 (11-9) 6-4.
Sixth seed Benoit Paire and fellow Frenchman Jeremy Chardy (13) recorded respective straight-sets wins over Teymuraz Gabashvili and Victor Estrella Burgos.
In first-round action, Marcel Granollers booked a tie with in-form countryman Rafael Nadal - last week's Monte Carlo Masters champion and an eight-time winner in Barcelona - by beating another Spaniard, Daniel Munoz de la Nava, 6-4 6-3.
Mikhail Youzhny safely negotiated his way into round two with a 6-2 6-2 beating of Rajeev Ram, while qualifier Marton Fucsovics upset the odds to beat Ernests Gulbis in three sets and there were also wins for Ivan Dodig, Pablo Carreno Busta, Thiemo de Bakker and Albert Ramos-Vinolas.
De Bakker will be the first opponent for two-time reigning champion Kei Nishikori.