OBJECTIVE To compare urine ligase and polymerase chain reaction (LCR, PCR) tests for diagnosis of Chlamydia trachomatis cervical infection with PCR and nucleic acid probe (GPA) on cervical specimens in adolescents, as well as risk factors for C. trachomatis infection and prevalence of infection at enrollment. METHODS Urine and cervical specimens were collected from women aged 13-20 years attending adolescent clinics, and interviews were administered. Urine specimens were tested by PCR and LCR, and cervical specimens by GPA and PCR. Prevalence rates of C. trachomatis infection and gonorrhea were compared by demographic, behavioral, and clinical risk factors. RESULTS Of 415 women tested, 86 (20.7%) were infected with C. trachomatis as indicated by positive cervical PCR results. A higher prevalence of C. trachomatis infection was seen among adolescents who douched monthly or more frequently, or had gonorrhea; prevalence declined from 25.8% in the first 7 months to 16.3% in the last 14 months of the study (p = .017). A statistically significant protective effect for reported condom use was not observed. Sensitivity of urine PCR was 89.5% and specificity was 100% relative to cervical PCR, compared to 84.9% and 99.4% (urine LCR) and 65.4% and 98.0% (cervical GPA). Sensitivity of urine PCR was higher in women with discharge; urine LCR sensitivity was higher in women < 19 years of age. CONCLUSIONS Polymerase chain reaction and LCR assays on urine specimens were sensitive, specific, and noninvasive tests in this population of adolescents with high C. trachomatis infection prevalence. Chlamydia trachomatis infection was associated with douching monthly or more frequently. Prevalence of infection declined over the period during which the study was conducted.