Detection of Chlamydia trachomatis infection was evaluated by culture and a new Syva enzyme immunoassay (EIA) in 1,012 patients at two Baltimore, Md., sexually transmitted disease clinics. The overall chlamydia prevalence determined by culture was 12%. For 506 fresh cervical and urethral specimens, the sensitivity of Syva EIA was 90% and its specificity was 94% compared with culture. Discordant Syva EIA results were further evaluated by staining the sediment in centrifuged culture transport media and Syva EIA transport tubes with a fluorescent monoclonal antibody to C. trachomatis to detect elementary bodies. Reanalysis of the data after use of this technique to resolve discordant results increased sensitivity and specificity to 92 and 96%, respectively. A subsample of 307 fresh cervical specimens was also tested in a three-way comparison using Abbott Chlamydiazyme, Syva EIA, and culture. In this sample, compared with culture, the sensitivity and specificity of Syva EIA were 87 and 95%, respectively, and for Chlamydiazyme they were 77 and 98%, respectively. Syva EIA is a 4-h, easy-to-perform enzyme-linked immunosorbent assay which has a high sensitivity with fresh genital specimens and offers an excellent alternative to culture.