Tenth seed Garbine Muguruza was knocked out of the US by Bulgaria's Tsvetana Pironkova - playing her first tournament in three years.
The 32-year-old notched a 7-5, 6-3 win - finishing it with a powerful ace.
"I'm really, really happy that I was able to win this match," said Pironkova afterwards.
She returns to tennis following a hiatus after having her son in April 2018.
"Coming here to the US Open, being my first tournament in three years, was a bit risky, but at the same time I was very excited and motivated to play well on this big stage, and I think that motivation kept me pushing."
Muguruza's frustration was evident, and she demonstrated that at the end of the first set as she smashed her racket on the ground.
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"I'm not a type of player that does that [smashing rackets] a lot," Muguruza told the media afterwards.
"I just feel like today out there was a tough first set. I felt I had it in control, then she played very well and took her chances. All of a sudden it was gone."