Background: The excellent sensitivity and specificity of commercially available nucleic acid amplification tests (NAATs) for the identification of Neisseria gonorrhoeae have been demonstrated. This study evaluated the performance of the BD ProbeTec ™ N. gonorrhoeae Q x (GCQ) Amplified DNA Assay on the BD Viper ™ System with XTR ™ Technology in a multicenter study. Methods: Specimens were collected at 7 geographically diverse clinical sites from 1846 women and men attending sexually transmitted disease, family planning, and obstetrics and gynecology clinics. There were 1768 evaluable participants, 994 women and 774 men. GCQ results from female endocervical, self-collected vaginal, male urethral swab specimens, and male and female neat (unpreserved) urine specimens, as well as those obtained using the urine preservative transport (UPT) tube for the GCQ assay were compared with patient infected status (PIS). For each participant, PIS was determined based on the combined results from the reference assays Aptima Combo 2 ® (AC2) and BD ProbeTec ™ ET GC Amplified DNA Assay (PT). Results: The sensitivity versus PIS for endocervical, vaginal, and female UPT urine, and female neat urine samples was 98.5%, 100.0%, 98.5%, and 96.9%, respectively; the specificity was 99.7%, 99.1%, 99.7%, and 99.5%, respectively. The sensitivity versus PIS for male urethral swabs and both male UPT and neat urine was 100.0%, with specificities of 99.1% for the urethral swab and UPT urine and 98.9% for the neat urine. The overall GCQ assay performance was not statistically different from that of AC2 or PT. Conclusions: The GCQ assay demonstrated performance characteristics comparable with other commercially available nucleic acid-based tests such as AC2 and PT. Vaginal swabs, endocervical swabs, urethral swabs, and urine specimens may all be used for gonorrhea screening. © 2012 American Sexually Transmitted Diseases Association All rights reserved.