Genitourinary infection with Chlamydia trachomatis is a common and potentially serious sexually transmitted disease. Diagnosis of C trachomatis infection in women typically relies on culture of endocervical swabs, an invasive and expensive procedure. The ligase chain reaction (LCR) is an in-vitro nucleic acid amplification technique that exponentially amplifies selected DNA sequences. We have compared an LCR-based assay to detect C trachomatis plasmid DNA in first void urine with culture of endocervical swabs for matched specimens from 1937 women from four geographic regions. Discordant specimen pairs were further tested by direct fluorescent antibody staining for elementary bodies and an alternative LCR assay based on the chlamydial outer membrane protein gene. An "expanded gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive women. The sensitivity and specificity of the LCR assay with first void urine samples compared with the expanded gold standard were 93.8% and 99.9%, respectively; the corresponding values for culture were 65.0% and 100%, respectively. Thus, an automated LCR assay of readily obtained urine samples showed a detection rate for infected women almost 30% greater than that of endocervical swab culture. The LCR assay was highly effective for the detection of C trachomatis in urine from women with or without signs or symptoms of chlamydial genitourinary tract infection.