In a desperate finish to the 100 freestyle sprint, Adrian got his fingers on the wall ahead of Australian world champion James Magnussen by 0.01 seconds, the closest margin to a dead-heat in Olympic swimming.
It was impossible to split the pair with the naked eye but Adrian, the first American to win swimming's premier event in nearly a quarter of a century, was awarded the gold in a time of 47.52 seconds with Magnussen second in 47.53.
Canada's Brent Hayden was third in a classic race where the eight finalists were separated by less than a second.
"I almost started crying in the water. This is something that happens every four years," said Adrian, the first American to win the event since Matt Biondi in 1988.
"It's not who swims the fastest time this year but it's who can get their hands on the wall first here tonight."
Magnussen went into the Olympics as the favourite after winning the world title last year and boasting that he would win but said he had learnt a painful lesson.
"I just felt pretty much bullet proof coming into this Olympics and it is very humbling," he said.
"They say what doesn't kill you makes you stronger so hopefully I can come out of this a better swimmer but most of all a better person."
Later on, Ryan Lochte, conserving his energy for one of the most brutal challenges in Olympic swimming, safely navigated his way into the final of the 200 metres backstroke.
Lochte won his semi-final in a time of one minute, 55.40 seconds to qualify for Thursday's final second fastest overall behind his American team mate Tyler Clary.
Clary won his semi-final in 1:54.71, nearly seven tenths of a second ahead of Lochte but knowing he will have to be even faster in the final to hold off his team mate.
"The swim went really well. I hit most of the things I wanted to hit," Clary said.
"There are a couple of things I want to work on for tomorrow, namely a couple of my turns and my breakout. I think tomorrow is going to be a pretty good swim."
Lochte is the Olympic champion in the event and the clear favourite to win the gold but faces an extra challenge because he is swimming the 200 individual medley against Michael Phelps on the same day.
China's Zhang Fenglin was third fastest overall in the backstroke semi-finals, just ahead of Japan's Ryosuke Irie, who was runner-up to Lochte at last year's world championships.
Phelps and Lochte both safely qualified for the final of the men's 200 metres individual medley.
The two Americans set up one last tantalising clash before Phelps retires after the Games when they navigated their way through the semi-finals and into Thursday's medal race.
Phelps, who won the 200 medley at Athens in 2004 and Beijing four years later, is bidding to become the first man to win the same individual event at three Olympics.
He beat Lochte at the U.S. trials but finished second behind him in the semi-finals and third overall in a time of one minute, 57.11 although both men will go much faster in the final.
"We love racing against each other," Phelps said. "Neither one of us likes to lose. I like to say we bring out the best in one another."
Lochte was fastest overall in 1:56.13 despite conserving his energy because he swam the 200 backstroke semi-finals just before the medley.
The second fastest overall time was set by Laszlo Cseh of Hungary, who left nothing to chance after missing out on a place on the final for the 400 medley.
"I have a good chance of winning a medal, but there are two good Americans in there," Cseh said.
South Africa's Chad le Clos dead-heated with Britain's James Goddard for seventh. Le Clos upset Phelps in the 200 butterfly final on Tuesday and was so shocked by his victory that he slept with his gold medal.
"I really didn't think I was gonna make it," he said.
"Obviously I'll fight to get that medal, but it will be difficult, a lot more difficult than last night."
FINAL STANDINGS - 100m men's freestyle:
1. Nathan Adrian (US) 47.52
2. James Magnussen (Australia) 47.53
3. Brent Hayden (Canada) 47.80
4. Yannick Agnel (France) 47.84
5. Sebastiaan Verschuren (Netherlands) 47.88
6. Cesar Cielo (Brazil) 47.92
7. Hanser Garcia (Cuba) 48.04
8. Nikita Lobintsev (Russia) 48.44
- James Magnussen
- Ryan Lochte
- Brent Hayden
- Michael Phelps