Veteran home favourite Venus Williams saw her Western & Southern Open campaign ended at the first hurdle as she lost in three sets to Lucie Safarova.
The seven time grand slam winner took the first set on a tie-break but could not maintain momentum and went down to the 16th seed 6-7 (2/7) 6-3 6-4 in Cincinnati.
Sarafova will next face qualifier Zarina Diyas from Kazakhstan who beat Irina-Camelia Begu 7-6 (7/4) 7-6 (7/5).
There was better news for another American as Sloane Stephens overcame Andrea Petkovic 6-3 6-3.
Maria Sharapova had been handed a first-round bye and advanced to the third round with a three-set win over Madison Keys.
The fifth seed eased through the first set but let slip the second to her American opponent before winning 6-1 3-6 6-3.
Also joining the Russian in the third round are second seed Simona Halep - a straight sets winner against Kirsten Flipkens - and Caroline Wozniacki, who eased past Shuai Zhang 6-2 6-3.
Lucky loser Mona Barthel failed to take take advantage of her reprieve as she suffered a first-round exit at the hands of Barbora Zahlavova Strycova.
The German had replaced defending champion Victoria Azarenka, who was forced to pull out through injury, but suffered a 7-5 6-0 defeat to her Czech opponent.
Carla Suarez Navarro advanced at the expense of Tsvetana Pironkova, 6-3 1-6 6-2, while Christina McHale beat Nicole Gibbs 4-6 6-3 6-1 in their all-American clash and Kurumi Nara was a 6-0 6-2 winner against Romina Oprandi.
Ekaterina Makarova was a comfortable winner over Daniela Hantuchova as she progressed 6-2 6-2 but 14th seed Sara Errani was made to work hard to beat Yanina Wickmayer 7-5 3-6 7-6 (7/2).
Svetlana Kuznetsova needed three sets to overcome Camila Giorgi 6-4 6-7 (7/9) 6-3 while Annika Beck beat Garbine Muguruza 6-3 2-6 6-3 and Sabine Lisicki was a 6-3 2-6 6-2 winner over Roberta Vinci.