John Isner was toppled by Australian Matthew Ebden to continue the American misery on the opening day of the Australian Open on Monday.
The 16th seed, playing in his 100th Grand Slam match, bowed out 6-4, 3-6, 6-3, 6-3 in just over two hours on Margaret Court Arena.
It was Isner's first defeat to Ebden in four meetings and follows the shock exits of Americans Venus Williams, US Open champion Sloane Stephens and last year's semi-finalist CoCo Vandeweghe in the women's draw.
It is the third time 78th-ranked Ebden has reached the second round at his home major and Isner was the first top-ranked 20 player he had beaten at a Grand Slam.
"It's unbelievable," Ebden said. "I've lost to John the last two times and he's so tough to beat but I got immense positivity from the crowd and I feel great and full of energy."
Ebden, the second-highest ranked Australian behind Nick Kyrgios, will face Ukraine's Alexandr Dolgopolov in the second round.