Five equine-virulent strains of Venezuelan encephalitis (VE) virus consistently killed guinea pigs (strain English short hair) inoculated subcutaneously, whereas ten equinebenign strains did not. Two enzootic eastern Panamanian and Colombian strains of hemagglutination-inhibition antigenic subtype I-D and of undetermined virulence for equine animals also killed guinea pigs. This subtype of VE virus may represent an exception to a positive correlation between equine virulence and guinea pig lethality since another strain of this subtype has been reported to be benign in horses inoculated subcutaneously. Within the strains tested, viruses of subtypes I-ABC and I-D were lethal for guinea pigs, and viruses of other subtypes were benign. There was no correlation between guinea pig and human virulence of VE strains.