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We have previously shown that a hydrophobic alkylating compound, 1,5-iodonaphthyl-azide (INA) can efficiently inactivate the virulent strain of Venezuelan equine encephalitis virus (VEEV), V3000 in vitro. In this study, we have evaluated the safety of INA-inactivated V3000 and V3526 and the protective efficacy of INA-inactivated V3000. INA-inactivated V3000 and V3526 did not cause disease in suckling mice. RNA isolated from the INA-inactivated V3000 and V3526 was also not infectious. Immunization of adult mice with INA-inactivated V3000 induced an anti-VEEV antibody response and protected mice from virulent VEEV challenge. The protective efficacy of INA-inactivated V3000 increased with the use of adjuvants. Results suggest that inactivation of enveloped viruses by INA may occur by two independent mechanisms and the INA-inactivated VEEV elicit a protective antibody response in mice.