The Roche cobas® CT/NG test (c4800), performed on the cobas 4800 system, is a new diagnostic assay using an automated workstation to isolate nucleic acids from clinical specimens and a real-time instrument for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). This study compared the performance characteristics of the c4800 with the Becton Dickinson ProbeTec™ CT/GC Q(x) assay (Q(x)) and Gen-Probe® Aptima Combo 2 (AC2) assay for the detection of CT and NG in male urine using patient-infected-status (PIS). Urine and urethral swabs were obtained from men attending STD, family planning, or OB/GYN clinics from 11 geographically distinct locations. Aliquot order was randomized for urine specimens between AC2, c4800, and Q(x). Urethral swabs were randomized between AC2 and Q(x). Urethral swabs were only used to define PIS and were not tested on the c4800. A participant was considered infected if the 2 comparator assays with different molecular targets had positive results from either sample type. A total of 790 men were screened, with 768 evaluable for CT and NG. Symptoms were reported in 296 (38.5%) participants. For urine, the overall sensitivity and specificity of the c4800 assay for CT were 97.6% and 99.5%, respectively, when compared with PIS. Sensitivity and specificity for NG were 100% and 99.7%, respectively. The c4800 has excellent sensitivity and specificity for male urine specimens when compared with PIS. Assay performance was similar in symptomatic and asymptomatic men and was equivalent to nucleic acid amplification tests that are currently on the market.