Enzyme-linked immunosorbent assays (ELISAs) were developed for detection of immunoglobulin G (IgG) and IgM antibodies to Venezuelan equine encephalomyelitis (VEE) virus in vaccinated and naturally infected humans. A total of 441 sera found negative for VEE antibodies by plaque reduction neutralization were examined by IgG ELISA and gave a 1.0% false-positive rate; no false-positives were found in the IgM ELISA. Sera with neutralizing antibody to western or eastern equine encephalomyelitis virus did not react with VEE antigen in the IgG ELISA. Sensitivity of the IgG ELISA was determined by testing 100 coded pre- and postvaccination human sera. Sixty-two were positive by ELISA; 58 of these 62 were also positive by neutralization tests, and 38 were negative by both tests. No neutralization-positive, ELISA-negative sera were found. Comparison of titers obtained by ELISA and neutralization tests indicated that 88% varied randomly by a fourfold dilution factor or less, while 61% were identical or varied only twofold. In sera obtained sequentially from 10 vaccinees and 5 naturally infected patients, both IgG and IgM antibodies appeared between 2 and 3 weeks after vaccination or onset of symptoms. The IgG and IgM antibody ELISAs described are rapid, specific, and sensitive indicators of VEE antibody status in vaccinated and naturally infected individuals.