A newly developed microwell enzyme immunosorbent assay (EIA) system by Syva Company (Palo Alto, CA) can detect Chlamydia trachomatis in < 3 hr. It uses a polyclonal antibody to chlamydial lipopolysaccharide and end points are determined with a spectrophotometer. Three clinical trial sites (University of California Medical Center, San Francisco, CA; University of Washington, Seattle, WA; and Louisiana State University Medical Center, New Orleans, LA), compared this EIA with tissue culture (TC) for identifying Chlamydia in urogenital specimens. Overall prevalence by TC was 10.4% (136 of 1306). When tests were compared with TC (using vials or microtiter plates and a fluorescent antibody stain), we found an EIA sensitivity of 93.4% (127 of 136) and a specificity of 98.1% (1148 of 1170). This EIA has a performance profile that is, at the very least, comparable with other nonculture methods for diagnosing genital tract infections with C. trachomatis.