Two strains of recently isolated Venezuelan equine encephalitis (VEE) complex virus from southern Brazil, avirulent for 6- to 8-week-old mice and short-haired guinea pigs, were characterized by biologic, serologic, and biochemical means. They were shown serologically to represent a single, newly recognized variant of subtype I. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of ribonuclease T1 digests of viral ribonucleic acid showed considerable homology between the genomes of the new variant prototype and variant IA. Three structural proteins were visualized by discontinuous sodium dodecyl sulfate-PAGE (SDS-PAGE). Although the smallest protein of both recent isolates migrates with the capsid proteins of other subtype I viruses, the larger structural proteins of the new variants differ in molecular weight from the E1 and E2 envelope glycoproteins of the other subtype I variants. The new isolates produced peptide fragment patterns that were identical to each other, but different from the patterns of other subtype I viruses, following SDS-PaGE of dissociated virions digested with Staphylococcus aureus V8 protease. Since these two isolates were from Culex (Melanoconion) species mosquitoes and from a bat (Carollia perspicillata), were postulated that this is an enzootic VEE virus variant for which the classification IF is suggested.