To evaluate the AntigEnz Chlamydia enzyme immunoassay (EIA) (Baxter/Bartels, Issaquah, Wash.), we studied 320 men and 1,209 women attending clinics for sexually transmitted diseases in Baltimore, San Francisco, and Seattle. At examination, two separate swabs were obtained from each patient, one for chlamydial culture and one for EIA. Cervical samples were collected from women, and urethral samples were collected from men. The prevalence of chlamydial infection by culture was 9% in Baltimore (n = 532), 11% in Seattle (n = 500), and 9% in San Francisco (n = 497). To resolve specimens with discrepant culture and EIA results, the EIA transport buffer was centrifuged and the resuspended pellet was stained by direct immunofluorescence to determine whether elementary bodies were present. Overall sensitivity of the AntigEnz Chlamydia assay compared with culture was 87% in men and 86% in women, and overall specificities were 94 and 97%, respectively. Differences between centers were seen, with sensitivities ranging from 76% among men and 79% among women in Seattle to 100% among men and 95% among women in Baltimore. With a true positive considered to be either a culture-positive or an EIA- and direct immunofluorescence-positive specimen, the revised sensitivity was 91% in men and 88% in women. Overall revised specificity was 99% in both men and women. We conclude that in this high-prevalence population, the sensitivity and specificity of this assay compare favorably with those of other noncultural antigen detection tests for the diagnosis of chlamydial genital infection.