The spacecraft blasted off as scheduled at 10:39 a.m. ET, following days of troubleshooting to fix a faulty fuel sensor in its external tank.
A fuel sensor problem that scrubbed the shuttle's liftoff nearly two weeks ago did not recur Tuesday, NASA said.
Discovery crew members, who awoke after midnight, appeared at a traditional photo opportunity wearing matching Hawaiian shirts in the crew dining room as astronaut Steve Robinson strummed a guitar. Later the crew donned space suits for their journey and, by 8:22 a.m. ET, all seven were aboard and strapped in.
Commander Eileen Collins was the first to board, giving a confident wave to NASA cameras before entering the cockpit.
Japanese astronaut Soichi Noguchi held up signs saying: "Get out of quarantine free" and "OUT TO LAUNCH."
At 9 a.m. ET, the hatch was closed.
About 10 miles from launch pad 39B, scores of shuttle-watchers who had camped out overnight along the Banana River were waking up excited about the liftoff.
"We were here when they tried to launch the first time," said Kai Novak, 41, of Hamburg, Germany. "When it didn't go, we were all really depressed. We're looking forward to seeing this today."
Also among the crowd was Thor Hurlen, 46, of Aalesund, Norway. "I think it's going to happen today," he said.
On July 13, NASA scrubbed Discovery's launch just 2 1/2 hours before liftoff when a test showed one of Discovery's four sensors in its hydrogen fuel tank was not working.
On Monday, after 12 days of troubleshooting, officials said they were again ready for liftoff but still weren't sure what caused the problem.
"We don't completely [know] because it looks like a grounding issue," said Discovery vehicle manager Scott Thurston.
An array of cameras was positioned to videotape the launch.
The cameras are among many new safety measures implemented after the Columbia disaster. They include cameras aboard two WB-57 aircraft that took off from nearby Patrick Air Force Base, according to a base spokesman. The planes are equipped with a video recording system that can capture visible and infrared images to look for any damage to Discovery during liftoff.
Columbia disintegrated on re-entry when super-heated gases entered the spacecraft through a hole created during liftoff by a falling piece of insulation foam, according to an investigative panel. All seven crew members died.
Discovery's crew is scheduled to test a battery of tools and techniques that NASA engineers developed after the loss of Columbia to inspect the spacecraft's heat-resistant exterior tiles for any damage that might occur during liftoff.
Discovery's mission also takes it to the international space station to deliver supplies and conduct repairs.
First lady Laura Bush and several members of Congress watched Tuesday's launch from a VIP observation area.
The mission is scheduled to last 12 days.
Here's to the crew of the Space Shuttle Discovery...