The performance of a commercial transcription-mediated amplification (TMA) assay (Gen-Probe Amplified Chlamydia trachomatis Assay; Gen-Probe, USA) was compared with that of the Roche Cobas Amplicor CT/NG polymerase chain reaction (PCR) (F. Hoffmann-La Roche, Switzerland) for detection of Chlamydia trachomatis in urine specimens. First-void urine specimens were collected from 658 patients: 320 men and 338 women. The results of the two tests were identical for 650 (98.8%) of the 658 patients. Among them, the test results were uniformly positive for 72 patients: 39 men and 33 women. In the cases in which the initial results of TMA assay and PCR were discrepant the specimens were reanalyzed and tested by an alternative TMA assay and a major outer membrane protein-based PCR. After analysis of discrepant results, 74 specimens (11.2%) were considered positive. All positive results were correctly identified by TMA assay, whereas PCR gave two false-negative and six false-positive results.