Common methods used to decontaminate and disinfect laboratory animal areas are difficult to standardize, labor-intensive, and potentially hazardous for staff members and the environment. As an alternative to traditional methods, we tested fumigation with vaporized hydrogen peroxide by using the VHP 1000 Biodecontamination System. The design of our air-conditioning system allowed the connection of the generator to any animal room by using the ventilation piping, thus forming a closed circuit. A 3-h cycle consisting of dehumidification, conditioning, sterilization, and aeration was developed and shown to be effective. The biodecontamination process was monitored during five independent trials using chemical and biological (Bacillus stearothermophilus spores) indicators. Contact plates for testing surfaces and room air for environmental bacteria, yeasts, and fungi consistently showed fewer than 10 colony-forming units per 100 cm2 or per 1 liter air. In addition, this method proved successful with heat-sensitive equipment like the blower units of individually ventilated caging systems. Overall, the system was easy to use and very effective in biodecontaminating animal rooms and equipment in a reproducible manner. There were no signs of corrosion or functional damage after more than 10 fumigation cycles. Work load and potential health risk for staff members and the environment was negligible.