Screening for chlamydial and gonococcal infection has been strongly recommended for all sexually active women under the age of 26. Advances in the ability to detect infection by nucleic acid detection techniques have improved access to screening methods in routine clinical practices. To meet the increasing demand for testing, a high-throughput system is desirable. We evaluated the performance of the Hybrid Capture 2 CT/GC (HC2) assay with the Digene Rapid Capture System (HC2-RCS). The results of HC2-RCS for endocervical samples from 330 women were compared to those of culture and the COBAS Amplicor PCR. For detection of chlamydial infection, HC2-RCS had a sensitivity and a specificity similar to those of PCR (P > 0.5) and an improved sensitivity compared to that of culture (P = 0.007). For identification of gonococcal infections, all assays performed similarly (P > 0.5). The performance of HC2-RCS was also compared to that of the manual HC2 format (HC2-M) with these samples and with 911 endocervical samples collected previously. The performance of HC2-RCS was equivalent to that of HC2-M; the overall concordance rates for the detection of chlamydia and gonorrhea were 99.7% (kappa = 0.97) and 99.8% (kappa = 0.97), respectively. When the HC2 assay was performed with a semiautomated system application designed for high throughput, it demonstrated high sensitivity and a high specificity for detection of both Chlamydia trachomatis and Neisseria gonorrhoeae.