Detection of Chlamydia trachomatis in specimens of urine from men with acute urethritis by means of a rapid and convenient polymerase chain reaction (PCR)-based kit is reported. Results of the PCR were compared with those obtained by an enzyme immunoassay (EIA). Of 52 first-voided urine specimens, 22 (42.3%) were positive for C trachomatis, as determined by both PCR and EIA; 4 (7.7%) were positive by PCR and negative by EIA, and only 1 (1.9%) was negative by PCR and positive by EIA. The remaining 25 (48.1%) were negative by both PCR and EIA. These findings suggest that PCR assay is suitable for detection of C trachomatis in urine and may be more sensitive than EIA. Further studies are indicated.