Summary: A simple and rapid procedure for the preparation of inactivated virus vaccine is described using β-propiolactone (BPL), an agent not previously employed in the preparation of vaccines. β-propiolactone is capable of inactivating viruses in 10–15 minutes at 37°C whereas formalin and phenol require days for the same degree of inactivation under parallel conditions. Satisfactory inactivated vaccines can be prepared with eastern equine encephalomyelitis virus, rabies virus and MM-virus with the use of BPL. A significantly higher degree of antigenicity is demonstrated with β-propiolactone-inactivated virus vaccines than with formalin- and phenol-inactivated virus vaccines. Two to four times the minimal effective viricidal concentration of BPL can be used in the preparation of virus vaccines without critical loss in antigenicity.