Wearing masks as a precaution, astronauts opened the hatch between the Russian and US section at 3:05 pm EST (2005 GMT), about 11 hours after an alarm raised concerns about a possible leak of the toxic substance.
"No indication of ammonia," said NASA mission commentator Rob Navias. "Crew doffed their masks and are getting about their evening business."
US station commander Butch Wilmore, NASA flight engineer Terry Virts and Samantha Cristoforetti, a flight engineer with the European Space Agency, left the US side at around 4 am (0900 GMT) and joined their three Russian colleagues in the Russian half of the orbital outpost, a research laboratory that flies about 260 miles (418 km) above Earth.
Mike Suffredini, the station program manager in Houston, said the evacuation reflected initial concerns that ammonia may have escaped from one of two cooling loops used to dissipate heat from water lines.
"There was never any risk to the crew," he added.
After the alarm, the astronauts put on masks, moved to the Russian side and closed hatches between the modules. Later they returned to the US lab, but evacuated a second time after rising air pressure indicated a threat might still be present.
Later analysis showed a computer glitch triggered several false readings, tricking the station's software into setting off the alarm, said NASA associate administrator Bill Gerstenmaier.
The crew was about two hours into their workday when they retreated to the Russian section. They had planned to spend Wednesday unpacking a Space Exploration Technologies (SpaceX) Dragon cargo capsule that arrived on Monday and setting up new scientific experiments.
The station, staffed by rotating crews of six astronauts and cosmonauts, is a project involving 15 nations, led by the United States and Russia. It has been permanently occupied since November 2000.