OBJECTIVE To compare first-voided urine specimens with paired endocervical swab specimens from women to determine the role of urine in complementing or replacing swab specimens for the detection of the chlamydial antigen. METHODS For 18 months, both endocervical swab specimens (the criterion standard) and urine specimens were tested for the chlamydial antigen, using an enzyme-linked immunosorbent assay (Chlamydiazyme, Abbott Laboratories, North Chicago, Ill). Positive results were confirmed using a blocking reagent (Abbott Laboratories) and/or a direct fluorescent antibody test (Micro-Trak, Syva, Palo Alto, Calif). A low level of chlamydial antigen (below the enzyme-linked immunosorbent assay threshold recommended by the manufacturer) was also looked for and, when found, was confirmed by the direct fluorescent antibody test. METHODS Prenatal and family practice clinics in a 500-bed community hospital. METHODS Specimens were collected from 489 random asymptomatic or symptomatic women. METHODS The detection of the chlamydial antigen from endocervical swab specimens was compared with the detection from first-voided urine specimens. RESULTS Acceptable swab and urine specimens were obtained from 300 (61.3%) of the patients. The antigen of Chlamydia trachomatis was confirmed in 20 (6.7%) of the 300 women. Of the infected patients, the antigen was detected in both swab and urine specimens for nine patients (45%), only in the swab specimens for eight (40%), and only in the urine specimens for three (15%). Testing urine in addition to endocervical swab specimens allowed for the detection of 18% more chlamydial infections, whereas confirming the presence of the antigen below the enzyme-linked immunosorbent assay cutoff resulted in the detection of 54% more infections. CONCLUSIONS Collecting multiple specimens and testing for low levels of chlamydial antigen may significantly improve the detection of chlamydial infections in women. First-voided urine may be an appropriate complementary specimen to endocervical swab specimens, but urine by itself does not allow for the adequate detection of the chlamydial antigen in women.