First-void urine specimens from 683 men (592 without symptoms) were tested for Chlamydia trachomatis by a polymerase chain reaction (PCR) with KL1 and KL2 plasmid primers and by a Chlamydiazyme enzyme immunoassay (EIA). Thirty-seven specimens were confirmed to be positive by using the EIA blocking reagent and a second set of plasmid primers (T1 and T2). By comparing unconfirmed PCR results (KL1 and KL2 primers only) with the blocked Chlamydiazyme EIA results, the sensitivity and specificity of PCR were 100% (37 of 37 specimens) and 99.5% (643 of 646 specimens), respectively. Three additional specimens were negative by EIA but positive by PCR and were confirmed to be positive with primers T1 and T2. Two of the three specimens were from men with symptoms. The confirmatory PCR assay performed equally well in detecting positive specimens from symptomatic (31 of 31) and asymptomatic (9 of 9) men. Comparison of confirmatory testing of first-void urine specimens by PCR and EIA showed that PCR was 100% sensitive (40 of 40 specimens) and that the EIA was 92.5% sensitive (37 of 40 specimens) but that the assays were equally specific (100%).