Molecular biology-based amplification methods are significantly more sensitive than other methods for the detection of Chlamydia trachomatis. The performance characteristics of the new Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay (AMP CT) with endocervical and urine specimens were compared to those of culture for patients attending two Baltimore City sexually transmitted disease clinics and a clinic for adolescents. AMP CT uses transcription-mediated amplification (TMA) and hybridization protection assay procedures to qualitatively detect C. trachomatis by targeting a 23S rRNA. Discrepant results between culture-negative and AMP CT-positive specimens were resolved by direct fluorescent-antibody staining of sedimented culture transport medium for elementary bodies and by TMA with 16S rRNA as a target. Following discrepant analysis, for 480 female urine specimens AMP CT had a sensitivity of 93.8% and a specificity of 100%. For 464 male urine specimens, the resolved sensitivity and specificity of AMP CT were 95.6 and 98.7%, respectively. For the 479 endocervical swab specimens the sensitivity of AMP CT was 100% and the specificity was 99.5%. Resolved culture sensitivities of AMP CT for female and male swab specimens were 52.3 and 58.9%, respectively. These results demonstrate that AMP CT is highly sensitive for the detection of C. trachomatis in endocervical specimens and in urine specimens from men and women.