We have prepared a murine monoclonal antibody (MAb) capable of distinguishing between wild-type Venezuelan equine encephalomyelitis (VEE) virus and the TC-83 vaccine derivative. This MAb, 1A2B-10, was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey VEE virus. The MAb reacts with prototype viruses from all naturally occurring VEE subtypes except subtype 6 in an enzyme-linked immunosorbent assay. It does not react with TC-83 virus or members of the western and eastern equine encephalitis virus complex or with Semliki Forest virus. This antibody will also differentiate between TC-83 and Trinidad donkey VEE virus in indirect immunofluorescence assays with virus-infected Vero cells.