The Abbott LCx ligase chain reaction (LCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae was evaluated by using swab and urine specimens from 562 patients. C. trachomatis results by LCR were compared to those by the Gen-Probe PACE 2 assay, whereas N. gonorrhoeae results by LCR were compared to those by culture. The Gen-Probe and LCR assays were performed according to the manufacturers' instructions. Gram-negative diplococci growing on modified Thayer-Martin medium were confirmed as N. gonorrhoeae by the GonoGen II assay. Supplemental data analysis was performed by major outer membrane protein PCR for C. trachomatis and probes for pilin gene detection for N. gonorrhoeae. A true-positive result for each pathogen was defined as a positive result for all three or two of three assays. Overall agreement among the six assays was 94.8%. C. trachomatis prevalence was 16.2%; N. gonorrhoeae prevalence was 5.5%. The overall sensitivity and specificity, respectively, for each test (after supplemental data analysis) were as follows: for C. trachomatis, Gen-Probe, 65.9 and 100%; LCR on urine, 90.1 and 100%; LCR on swab specimens, 96.7 and 100%; and for N. gonorrhoeae, culture, 80.6 and 100%; LCR on urine, 93.5 and 99.8%; and LCR on swab specimens, 96.8 and 100%. For women, the N. gonorrhoeae culture was very insensitive compared to its performance in men (58.3 versus 94.7%, respectively). For C. trachomatis, the Gen-Probe assay's sensitivity was lower for men than for women (62.3 versus 71.1%, respectively). The sensitivity for C. trachomatis detection by LCR on urethral and cervical swab specimens was 96.2 and 97.4% for men and women, respectively. For men, swab results were slightly better than urine results for both pathogens (sensitivity for C. trachomatis in swab and urine specimens, 96.2 and 92.5%, respectively; sensitivity for N. gonorrhoeae in swab and urine specimens, 100 and 94.7%, respectively), while for women, cervical swabs were superior in sensitivity to urine samples for detecting C. trachomatis (swab, 97.4%; urine, 81.6%) and equivalent for N. gonorrhoeae (swab, 92.3%; urine, 91.6%). The LCx LCR appears to be both sensitive and specific for the detection of C. trachomatis and N. gonorrhoeae when performed on urine or genital swab samples. Swab samples had better sensitivity than urine samples for the detection of both pathogens.