Trichomonas vaginalis is estimated to infect 4 million women per year in the United States. The diagnosis of trichomoniasis is predominantly achieved by direct microscopic examination of vaginal exudates. This subjective diagnostic procedure is reported to be 75% sensitive under ideal circumstances. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of T. vaginalis directly from vaginal exudates. The ELISA employs a monoclonal antibody specific for a 65-kilodalton surface polypeptide of T. vaginalis as the capture antibody in a sandwich format. A polyclonal rabbit anti-T. vaginalis antibody labeled with horseradish peroxidase serves as the probe. An evaluation of vaginal specimens from women attending clinics revealed a sensitivity and specificity of the ELISA of 89 and 97%, respectively, versus the culture technique. These results indicate the usefulness of this ELISA as an alternative to microscopic and culture methods for the detection of T. vaginalis in vaginal exudates.