To determine the accuracy of a recently developed polymerase chain reaction (PCR) urine assay to detect Chlamydia trachomatis urethral infection in men, we obtained urethral swabs and first-catch urine from 365 men attending a sexually transmitted diseases clinic. Thirty-three (9%) of the 365 men were infected with C. trachomatis as defined by urethral culture. Thirty-two of the 33 men with culture-positive urethral swabs also had PCR-positive urine assays. Of 332 patients with culture-negative urethral swabs, 325 had PCR-negative urine. Compared with chlamydia culture of urethral specimens, PCR assay of urine samples thus had a sensitivity of 97% and a specificity of 98%. The positive predictive value of the urine PCR assay was 82%, and the negative predictive value was 99%. Analysis of discrepant results indicated that six of seven PCR-positive, urethral culture-negative patients probably had chlamydial urethritis. All six patients had symptoms of urethritis and had either a positive urethral swab PCR or a positive urine PCR with a different amplification target. After resolution of discrepant results, (defining true positives as the 33 culture-positive patients and the 6 PCR-positive, culture-negative patients just described), the sensitivity and specificity of culture were 85% (33 of 39) and 100% (326 of 326), respectively. The revised sensitivity and specificity of PCR were 97% (38 of 39) and 99.7% (325 of 326), respectively. We conclude that this urine PCR assay provides a highly sensitive, noninvasive alternative method for the detection of C. trachomatis urethral infection in high-risk men attending a sexually transmitted diseases clinic. This assay could greatly facilitate the testing of larger numbers of male patients for chlamydial infection and should be studied in other settings.