Usain Bolt, the reigning Olympic gold medalist and world record holder in the 100-metre dash, finished a millisecond behind American Justin Gatlin at the Golden Gala event in Rome.
Bolt, 26, led out of the blocks but Gatlin, who claimed a bronze in the event at the 2012 London Olympics, crossed the line at 9.94 seconds.
"I got the perfect start that I wanted, then about five steps in I stumbled a bit and that kinda threw my game off," Bolt told BBC Sport following the race.
France's Jimmy Vicaut came third at 10.02. Bolt's countryman, London silver medalist Yohan Blake, did not participate Thursday.
Prior to a May 8 race in which he was also the runner-up, Bolt was sidelined by a hamstring injury. The Golden Gala was his second race of the season.