Great Britain's Dai Greene finished back in fourth as Felix Sanchez won a surprise 400m hurdles gold.
World champion Greene was 0.14 seconds off a medal as American Michael Tinsley took silver and favourite Javier Culson bronze.
Two weeks before his 35th birthday, Sanchez was considered years past his prime, but in winning here repeated Angelo Taylor's trick of grabbing a second Olympic gold eight years after his first.
Tired Dai Greene surprised by final pace
The veteran from the Dominican Republic went out hard, just as he had in his semi-final, and led off the final bend as Tinsley edged a tiring Culson.
Greene tried to close in over the final two barriers as he had in winning world gold in South Korea last summer, but despite wonderful support from another capacity crowd, could make only limited inroads, just pipping veteran American Taylor for fourth on the line.
His time of 48.24 seconds was still faster than his gold medal-winning run in Daegu, but with Sanchez clear in 47.63secs, Tinsley 0.33secs back and Culson clocking 48.10secs, it was not enough to get him on the podium.
Greene told BBC Sport: "I felt tired tonight. I gave it everything I had but I was too tired at the end and narrowly missed out.
"Dai Greene will be bitterly disappointed that he hasn't got a medal but I can tell you it was a very, very fast final. We know that Dai has suffered from injury problems which has hampered his preparations and I can't help thinking that maybe this race came two weeks too early for him."
"I ran a personal best a few weeks ago, but perhaps the endurance wasn't quite there for the rounds. You can't always know until you get to the majors.
"Coming in, me and Culson were way ahead in the rankings, but a few of the guys pulled something out. They're great competitors and Felix is a deserving winner."
Greene's personal best - set at the Paris Diamond League last month - would have been good enough for silver and an improvement of two-tenths of a second could have given him gold.
But after losing a large chunk of his early winter's training to a knee injury, he has been playing catch-up for much of the season, and that lack of endurance was obvious in the final straight.
Sanchez overwhelmed by gold medal
Sanchez's winning time was the same as the one that won him Olympic gold in Athens 2004. He dedicated victory to his late grandmother, having run with a photo of her pinned inside his vest.
Asked what the reaction would be back home in the Dominican Republic, Sanchez said: "It's going to be huge. They expected the Worlds [the titles he won in 2001 and 2003], but no-one expected this. A lot of people said I should retire but I stuck with it."
His victory is something of a fairytale story, not only for his age and second title but because less than a month ago he was trailing home fifth at the London Grand Prix in Crystal Palace, three places behind Greene.
The man who won in London that night, Culson, will take little succour from the fact that his bronze was the first Olympic medal Puerto Rico have ever won outside the boxing ring.
World number one all year, he had not lost outdoors this summer before these Olympics.
"I'm not pleased with it at all," he said. "I lost my rhythm towards the end of the race and I couldn't get it back. I send my apologies to the people back home, but at least I made it to the final."
But Tinsley, sixth at Crystal Palace and with a season's best a full four-tenths of a second slower than the Puerto Rican, produced a personal best performance when he most needed it.