A multicenter study compared ligase chain reaction (LCR) of Chlamydia trachomatis plasmid DNA with culture of urethral swab specimens from 542 men (study A); a second study (B) compared LCR of first-void urine (FVU) with urethral swab cultures from 1043 men. Discordant results were resolved with direct fluorescent antibody staining of sediments from the FVU or urethral culture specimen and with a second LCR directed against a fragment of the major outer membrane protein gene. Test performance was calculated on the basis of an expanded reference standard. The LCR plasmid assay had a sensitivity of 98.0% in study A and 93.5% in study B; specificity was 99.8%-100%. The sensitivity of culturing urethral swabs from all study sites was 68.2% (range by sites, 40.0%-84.6%). The presence or absence of urethral symptoms did not alter the results. Use of this LCR test should allow more meaningful investigation and treatment of C. trachomatis infections in men.