Gay won in 9.99 seconds after Gatlin failed to sustain a good start and finished with a time of 10.03 just three weeks before the start of the London Olympics. Eleven days ago Gay was beaten by Gatlin at the US trials.
"I took my revenge. Maybe I made some mistake at the start. I want to win the gold medal (in London)," said Gay.
Gatlin added: "The goal is the gold, my chances are good. I don't want to train to be second or third. I was a bit tired tonight. I could have done better".
Twice European champion Christophe Lemaitre of France took third place in 10.08 as light drizzle fell on the stadium.
Dejen Gebremeskel led an Ethiopian one-two in the 5,000 metres, clocking a world leading 12:46.81 to beat Hagos Gebrhiwet, who set a junior world record of 12:47.53.
Bekele, however, conceded he would not defend his title in London after being only the fifth Ethiopian to cross the line.
"I won't compete at the Olympics in the 5,000 metres but I'm not that disappointed because I'll run the 10,000 metres," he said.
Referring to the calf problems he has suffered for three years, he added: "What can I do about an injury?"
David Rudisha was one of five athletes to set a world season's best but the Kenyan failed in his attempt to break his own world record in the 800 metres, clocking 1:41.54, 0.53 outside the mark he set in 2010.
"I'm happy and I'm impressed with the way I ran today. The Olympic title is what I'm aiming for now," said Rudisha, who beat second-placed Antonio Manuel Reina of Spain by more than four seconds.
"I'll go back home to train and get ready for the Olympics and then I'll be looking for a fast race and the world record," he added.
Australian Sally Pearson was again in a class of her own when she destroyed the field in the women's 100 metres hurdles with a world leading 12.40 seconds.
"I was expecting a fast time and if I would have a clear race it could be a 12.3. I'm excited to see my shape," she said.
The world record of 12.21, held by Bulgarian Yordanka Donkova since 1988, is one of the oldest in athletics.
French pole vaulter Renaud Lavillenie, who is unbeaten outdoors this year and will be the hot favourite in the Olympics, only needed to clear 5.77 metres to win the event.
He then failed with three attempts at 5.82 metres.
"The incredible jump will have to come in London (at the Games)," he said.
Britain's Steve Lewis placed fourth
Puerto Rico's Javier Culson won the 400 metres hurdles in a world season's best time of 47.78 ahead of world champion David Greene of Britain.
Greene fractionally failed to rewrite Kriss Akabusi's British record with a storming personal best .
Last week hurdles legend Ed Moses questioned whether Greene was fast enough to add the Olympic crown to his collection of titles and he responded by clocking 47.84 seconds at the Stade de France, just 0.2 secs outside Akabusi's long-standing best time.
"It feels good to be back in business, all coming together at the right time. Ready for the big one," he wrote on Twitter.
Culson, however, does not see himself as favourite to win the Olympic race.
"I'm not considering myself as the Olympics favourite, I'm not the only one, there are other athletes, especially Greene who will be at home," he said.
Moroccan Mariem Alaoui Selsouli also set a world season's best when she won the 1,500 metres in 3:56.15. Lisa Dobriskey finished eighth.
Elsewhere, defending Olympic champion Christine Ohuruogu ran a season's best 50.59 secs to finish fourth over 400m behind Amantle Montsho while Shara Proctor settled for second behind Elena Sokolova in the long jump, with a best leap of 6.65m.
Barbara Parker finished eighth in the 3,000m steeplechase as Tunisia's Habiba Ghribi prevailed.