Urine samples from 467 men living in the Stockholm area were tested with the polymerase chain reaction (PCR), Roche Amplicor, and with an enzyme-linked immunosorbent assay, Syva MicroTrak EIA, for detection of Chlamydia trachomatis. The predictive value of urine versus urethral samples was subsequently compared on a second urethral sample from 25 C. trachomatis-positive cases. The urethral samples were in addition cultured for C. trachomatis. C. trachomatis was found more often in urine by Roche Amplicor than by Syva MicroTrak, 9.9% and 7.9%, respectively. Nine urine samples, positive only by Amplicor, could be confirmed as true positives by complementary testing. C. trachomatis was detected with the same frequency in urine and urethral samples. The sensitivity was highest for PCR, 88% and 92%, and lowest for EIA, 76% and 80%, on urethral and urine samples, respectively. Urine sampling, offering a non-invasive procedure, was found suitable for the diagnosis of C. trachomatis in men, with the use of Roche Amplicor.