The suitability of urine specimens from women and men for the detection of Chlamydia trachomatis infection by a ligase chain reaction (LCR)-based assay with plasmid primers was examined with a group of patients attending a sexually transmitted disease clinic in Amsterdam, The Netherlands. Cervical specimens from 15 of 237 (6.3%) women tested positive for C. trachomatis by cell culture. Of the 25 (10.5%) female urine samples that tested positive by the plasmid-LCR assay, 13 were obtained from cervical culture-positive women. Nine of the 12 plasmid-LCR-positive urine samples from cervical culture-negative women were confirmed to be positive by a second LCR assay with primers based on chromosomal DNA. Urethral specimens from 24 of 258 (9.3%) men were positive for C. trachomatis infection by cell culture. Of the 25 (9.7%) urine samples that tested positive by plasmid-LCR, 20 were from culture-positive men. All five of the LCR-positive urine samples from culture-negative men were confirmed to be positive by the LCR with chromosomal DNA primers. Relative to cell culture, testing by plasmid-LCR analysis of male urine samples had a sensitivity of 83.3% and a specificity of 97.9%; after resolution of discordant samples, these values were 86.2 and 100%, respectively. In the study with women, the sensitivities of plasmid-LCR analysis of cervical and urine specimens in comparison with cervical cell culture were 93.3 and 86.7%, respectively. After resolution of discrepant samples, the sensitivities of the plasmid-LCR test for cervical swabs and female urine samples were 96.3 and 92.6%, respectively. These results indicate that the plasmid-LCR-based assay is a very reliable, sensitive, convenient test for the detection of C. trachomatis infection in female and male urine specimens.