Top seed Sam Querrey exited the Hall of Fame Tennis Championships in the first round after being beaten in straight sets by fellow American Tim Smyczek.
Smyczek, ranked 99 places below his opponent at 120th in the world, broke once in the first set and then went on to edge a tight tie-break in the second to win 6-4 7-6 (10/8).
Third and fourth seeds Igor Sijsling and Lleyton Hewitt progressed safely to the second round of the Newport, Rhode Island event.
Dutchman Sijsling beat Italy's Matteo Viola 6-1 6-1, a scoreline mirrored by the veteran Hewitt against his fellow Australian Matthew Ebden.
However, the fifth, sixth and seventh seeds all exited the tournament.
Croatia's Ivo Karlovic accounted for Edouard Roger-Vasselin of France 7-6 (7/5) 6-4, Marinko Matosevic withdrew after losing the first set 6-2 to Jack Sock and Kenny De Schepper of France lost 6-3 6-4 to American qualifier Alex Kuznetsov.
Eighth seed Rajeev Ram beat Adrien Bossel 6-2 6-2 and Michael Russell was another American winner, 6-1 4-6 6-4 against Illya Marchenko of Ukraine.
Nicolas Mahut beat Rhyne Williams 6-2 6-4 and there were also victories for Jan Hernych, Prakash Amritraj and Vasek Pospisil.