Three monoclonal antibodies were generated that are specific for the E2 glycoprotein of Venezuelan equine encephalitis (VEE) virus and have useful reactivities in an enzyme-linked immunosorbent assay (ELISA). Antibody 1A1B-9 distinguished between the IC (epizootic) and ID (enzootic) varieties of VEE virus by ELISA. Clone 7A1A-1 antibody distinguished the Panamanian prototype virus (3880) from Colombian ID isolates by a 500-fold difference in titer by endpoint ELISA, and it detected antigenic variation in ID isolates from southern Colombia and Ecuador. Antibody 7A3A-4 defined a cryptic antigenic site on the latter two isolates. These monoclonal antibodies complement others in identifying VEE isolates by a simple ELISA.