We evaluated the Gen-Probe Chlamydia trachomatis transcription-mediated amplification (TMA) assay with urine specimens for the detection of C. trachomatis infections in women. The novel test, based on the isothermal amplification of chlamydial RNA, was compared with the Roche Amplicor PCR with urine and cell culture with endocervical specimens. First-catch urine and endocervical swab specimens were collected from a total of 561 patients, of whom 70 (12.3%) were confirmed to have chlamydial infection. The diagnostic sensitivity and specificity of TMA with urine were 91.4 and 99.6%, respectively, and those of Amplicor PCR were 97.1 and 99.8%, respectively. By repeated analysis of the specimens with discrepant results, the sensitivity of TMA could be increased to 99%, indicating that some methodological improvements in the assay are still to be expected. The sensitivity of PCR could be increased to 100% by the elimination of DNA polymerase inhibitors in a repeated analysis. The sensitivity and specificity of cell culture with cervical specimens were 85.7 and 100%, respectively. The results indicate that TMA with urine specimens from women is a sensitive and specific assay for the detection of C. trachomatis, providing a new noninvasive technique for the screening of chlamydial infections in women.