Gatlin upstaged the Jamaican with the top time in the first round, even though the American got a little push from nature with a strong back wind of 2.1 meters per second during his 9.83-second race.
Bolt could count on an adoring crowd but faced a slight headwind as he cruised to a time of 9.96, the fifth fastest overall.
"It was OK. It was not perfect," Bolt said. "I still have some adjustments. Just have to concentrate on my technique now."
Gatlin had no such complaints after his run.
"I felt safe, like, after 50 meters so I did not have to push it too much," said Gatlin, who was .13 seconds faster than Bolt.
Those results will mean nothing by Sunday's semifinals and final, when the crowd at the Bird's Nest will be hoping for a memorable head-to-head between the sport's biggest star and the 33-year-old American who has not been beaten in two seasons after returning from a second doping ban.
Bolt has been struggling with injury and form during Gatlin's comeback streak and on Saturday even his showboating was turned off. Beyond a few perfunctory waves and smiles, it was all business from the bearded Bolt. The flashiest part of his showing at the stadium where he won 100 gold with a world record seven years ago were his lemon-yellow colored shoes.
Gatlin, who served a four-year doping suspension midway through a career that started with a gold medal at the 2004 Athens Olympics and a sprint double at the 2005 worlds, faced some scattered boos from fans but nothing like the hostile media reception he has had in several nations.
He flaunted his form, though, looking left from the outside lane just past the midway mark and coasting from then on to set the top time.
Bolt, who celebrated his 29th birthday on Friday, was slow out of the blocks but once he was fully upright he used his massive strides to make victory look easy.
Even though they were only heats, the 100 dominated the opening day of the nine-day event. In the morning session on a sweltering but clear day, 19-year-old Ghirmay Ghebreslassie became the youngest man to win the marathon at the world championships and the first world champion from Eritrea.
Ghebreslassie finished in 2 hours, 12 minutes, 27 seconds, holding off Yemane Tsegay of Ethiopia by 40 seconds. Solomon Mutai of Uganda was third, while his teammate, defending world champion Stephen Kiprotich, was sixth.
The biggest surprise of all was the wipeout of the Kenyan team, which some favored to get a sweep in Beijing. Instead, world-record holder Dennis Kimetto and Kenyan teammate Wilson Kipsang dropped out of the race while Mark Korir finished 22nd.
Olympic 800 champion David Rudisha, one of the many great runners on the Kenyan team, led from start to finish with his majestic stride in full motion to reach the semifinals of his event.
And after the marathon setback, Rudisha was glad to see all three Kenyans advance in his event.
"This," Rudisha said, "is a boost for morale."