The highest-ranked American failed to convert a match point in the fourth set and lost in the first round to Alejandro Falla of Colombia, 6-4, 6-7 (7), 3-6, 7-6 (7), 7-5.
Isner is best known for winning the longest match in tennis history two years ago in the first round at Wimbledon, when he edged Nicolas Mahut 70-68 in the fifth set. Isner also went five sets in the second round at the French Open last month, losing 18-16 to Paul-Henri Mathieu. He has been eliminated in five sets in each of the past three Grand Slams.
Since climbing to a career-best No. 9 in the rankings in mid-April, Isner has gone 3-5.
"I'm just really down on myself right now," he said. "I'm trying not to feel the outside pressure. There are some good things expected of me, and I'm glad I'm in that position, but I'm just not performing right now. It's just ugly right now."
The 6-foot-9 Isner hit 31 aces and said he has been pleased with his serve, but not the rest of his game.
"Sometimes, and lately it has happening quite a lot, I get out there in the match and I'm just so clouded," he said. "I just can't seem to figure things out. I'm my own worst enemy out there. It's all mental for me, and it's pretty poor on my part."
Isner held a match point with Falla serving at 6-7 in the fourth set. The Colombian was on the ropes again serving at 5-all in the final set when he faced three break points, but Isner failed to convert them, then lost serve in the final game.
"I didn't put my opponent away," Isner said. "I had my chances, and I didn't do it. It's all on me."
American men went 3-3 on the first day of the tournament. Ryan Harrison won and will next face top-ranked Novak Djokovic, the defending champion. Michael Russell and Ryan Sweeting also advanced, while James Blake and Donald Young lost.
U.S. women went 2-3. Sloane Stephens and Jamie Lee Hampton won, while Venus Williams, Melanie Oudin and Vania King were eliminated.