Foot-and-mouth disease virus was inactivated with binary ethylenimine formed apart from or directly in the virus suspension by the cyclization of 2-bromoethylamine hydrobromide or 2-chloroethylamine hydrochloride under alkaline conditions. The inactivation rates with binary ethylenimine prepared apart from the virus suspension in dilute sodium hydroxide with either 2-bromoethylamine hydrobromide or 2-chlorethylamine hydrochloride were higher than with pure ethylenimine. When binary ethylenime was prepared directly in the virus suspension only 2-bromoethylamine hydrobromide gave acceptable inactivation rates. The reduced inactivation rates for binary ethylenimine directly prepared in the virus suspension are due to the different cyclization rates of 2-bromoethylamine hydrobromide and 2-chloroethylamine hydrochloride and to the interference of bicarbonate in the cyclization reaction. The complement fixing antigen of foot-and-mouth disease virus was not affected by binary ethylenimine inactivation. Vaccines prepared with foot-and-mouth disease virus inactivated by binary ethylenimine were comparable in their immunogenicity to vaccines prepared with ethylenimine or N-acetylethylenimine used as inactivants. Application of binary ethylenimine in the preparation of foot-and-mouth disease vaccines considerably reduces the potential danger associated with handling pure ethylenimine and other aziridines.