The high sensitivity of nucleic acid amplification tests such as ligase chain reaction (LCR) has the potential to simplify specimen collection for the microbiologic diagnosis of gonorrhea. We screened first-void urine specimens from 283 women attending a Birmingham, Ala., sexually transmitted disease (STD) clinic by using LCR and compared the results to those of cervical and urethral cultures for gonorrhea diagnosis. Fifty-three (18.7%) women had positive cervical cultures for gonorrhea, and 41 of the 53 (77%) also had positive urethral cultures. One additional patient had only a positive urethral culture (the cervical gonorrhea culture was negative). LCR testing of urine specimens for gonorrhea yielded positive results for 51 of 54 (94.4%) women with positive cervical or urethral cultures. Of 229 women with both urethral and cervical cultures negative for gonorrhea, 2 (0.8%) had positive urine LCR results as well. To resolve the discrepancies between urine LCR and culture results, LCR tests of simultaneously collected urethral and cervical swab specimens and LCR tests of the same urine specimens using different nucleotide primers were conducted. After evaluation of five discrepant results, the sensitivity, specificity, positive predictive value, and negative predictive value of LCR for the detection of gonorrhea in urine specimens were 94.6%, 100%, 100%, and 98.7%, respectively. We conclude that urine LCR testing for Neisseria gonorrhoeae is a practical alternative to culture for the detection of gonorrhea in women. Urine testing for STD diagnosis has the potential to simplify and expand the opportunities for STD screening and surveillance of women.