Butterflyer Ian Crocker dived in before Scott Usher completed his breaststroke leg, resulting in the DQ. It was the first time any U.S. relay team failed to reach a final in world championships' history.
"Just an unlucky exchange," said Neil Walker, who swam the anchor freestyle leg.
Ryan Lochte led off on the backstroke.
Crocker was back in the pool for the first time since losing the 100 fly to Phelps the night before. Phelps' victory was his sixth gold medal of the meet, tying Ian Thorpe's record from 2001.
Walker attributed Crocker's mistake to "a little bit of overexcitement."
"Ian had such an awesome race last night with Michael Phelps. To get back up the next morning is tough to do in the morning relay," he said.
Crocker completed the exchange in 0.04 seconds - one-hundredth of a second outside the allowable time, so no protest could be lodged.
"That's just incomprehensible, that amount of time," Walker said. "It's so small that you can never really plan that."
The result provoked instant reaction from other countries. Members of Singapore's relay pointed at the â€˜DSQ' listed next to the United States on a television monitor and smiled. Phelps was counting on his teammates to earn a spot in the evening final, where he would have joined the team, along with breaststroker Brendan Hansen.
"Michael was doing something that nobody has ever done before," Walker said. "I think everybody is going to be disappointed, Michael included. But he's going to see that this is the way it happens sometimes."
Walker spoke on behalf of the relay team, with Crocker walking by reporters in silence. Japan qualified first for the medley relay final in 3 minutes, 37.04 seconds. Russia was second and South Africa third.
Earlier Sunday, Phelps easily advanced to the 400 individual medley final, giving him a shot at a seventh gold. He qualified in 4:12.01 in a race where he owns the world record.
Going into Sunday night, Phelps had won four golds with the fastest times ever.
But a few other swimmers still harbored hopes of spoiling Phelps' last race in Melbourne. "A lot of guys are closing up on him," said Ous Mellouli of Tunisia, the 400 freestyle silver medalist and 800 free champion who qualified third. "I've never been that fast in the morning. I'm pretty confident." Italian Luca Marin, the boyfriend of French star Laure Manaudou, was second quickest. Lochte, who stunned Aaron Peirsol to win the 200 backstroke, was fourth. Laszlo Cseh of Hungary, the defending champion, was fifth. "It should be one of the most exciting races of the meet," said Mellouli, who is based in Southern California. American Katie Hoff, who trains at the same North Baltimore club that launched Phelps, led the women's 400 IM qualifying in 4:38.21. The defending champion was 1.91 seconds ahead of Jennifer Reilly of Australia. Hoff's teammate Ariana Kukors was fourth. Hoff is seeking her third gold medal, having won the 200 IM and anchored the victorious 800 free relay. She was fourth in the 200 free. "I feel really good. That was my best morning swim," she said. "I'm excited to finish off the meet strong. I've had almost all best times. I'm just very proud of how I've done." Kirsty Coventry of Zimbabwe, the silver medalist two years ago in Montreal, finished second to Hoff in their heat, but was disqualified for a one-handed touch at the end of the breaststroke leg. Swimmers are required to use both hands.
Entering the last day of the competition, the Americans had cornered the market: 17 golds and 31 medals overall in the temporary pool at Rod Laver Arena - not to mention 10 world records.
They were just one medal short of their performance at the 2005 worlds in Montreal, and still had an outside shot at chasing down the 1978 team as the most prolific hoarders of neckwear in world championship history.