The suitability of urine specimens for the detection of Chlamydia trachomatis infections in men was assessed. Urethral swabs from 301 patients were cultured for C. trachomatis, and the results were compared with results obtained from Chlamydiazyme. The results of 298 specimens were also compared with results obtained from PCR analysis of first-void urine specimens. The sensitivity of confirmed Chlamydiazyme analysis was 93% and the specificity was greater than 99% compared with culture. The sensitivity of the PCR method was 100% compared with culture. Chlamydia trachomatis was detected by PCR in an additional three specimens from which C. trachomatis could not be cultured. Urine appears to be an appropriate specimen for the detection of C. trachomatis antigens and nucleic acids.