Strains of Venezuelan encephalitis virus isolated from enzootic habitats during interepizootic periods in Middle America and northern South America can be distinguished from each other antigenically by hemagglutination inhibition. This test has provided the basis for the classification of these virus strains into subtypes I-E and I-D, respectively. Virus strains of these two subtypes have been found to differ profoundly with respect to virulence for English short hair guinea pigs. Studies are described which confirm that virus strains of the I-D subtype are guinea pig virulent, and that virulence is not the result of cocycling subpopulations of epizootic subtype I-AB or I-C virions. Two additional markers were found which distinguish subtype I-D and I-E Venezuelan encephalitis virus strains. Firstly, hydroxylapatite chromatography of intact virions at pH 6.5 showed differential elution of I-D and I-E prototype strains. Virions of subtype I-D strains eluted at 0.08 to 0.11 M phosphate, while those of subtype I-E strains eluted at 0.15 to 0.20 M phosphate. Secondly, the isoelectric points of the E1 envelope glycoproteins of the I-D and I-E prototype strains were significantly different; pH 6.85 to 7.00 and pH 7.25 to 7.30, respectively. There was no significant difference in the isoelectric points of the E2 envelope glycoproteins. These distinguishing characteristics most likely reflect a fundamental difference in virion surface structure.