There are currently no commercially available molecular assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in rectal swabs with regulatory approval. We compared the Cepheid GeneXpert CT/NG assay with the GenProbe Aptima Combo2 assay, using 409 rectal swabs. Using Aptima as the gold standard, the sensitivity, specificity, and positive and negative predictive values of GeneXpert for the detection of C. trachomatis and N. gonorrhoeae were 86%, 99.2%, 92.5%, and 98.4% and 91.1%, 100%, 100%, and 98.6%, respectively. Despite significant dilution of samples prior to GeneXpert testing, the assay performed well with excellent specificity. Chlamydia trachomatis and Neisseria gonorrhoeae are high-burden sexually transmitted infections (STIs) affecting significant numbers of the general population and high-risk subgroups. A total of 1,307,893 cases of chlamydia (426/100,000 population) and 309,341 cases of gonorrhea (100.8/100,000 population) were diagnosed in the United States in 2010 (3). The respective prevalences in England were 351 and 39.5 per 100,000 population, respectively, in 2011 (8). There has been a recent increase of 61% in cases of gonorrhea in men who have sex with men (MSM) in England. This could be explained by the increased use of nucleic acid amplification tests (NAATs) for extragenital samples (8). Management and control of these infections are heavily reliant upon highly sensitive and specific diagnostic techniques and tracing of sexual contacts.There is now a wide body of evidence confirming that NAATs are significantly more sensitive than culture for detection of genital STIs. These assays have become a standard diagnostic technique and are now recommended for testing genital and extragenital sample types by various bodies, including the Centers for Disease Control and Prevention (CDC) and the United Kingdom Health Protection Agency (HPA) (4, 7). In the United Kingdom, 82% of sexual health clinics offer asymptomatic MSM screening by NAATs for chlamydia, and 23% are offered NAATs for gonorrhea (2). Several studies using NAATs for the detection of N. gonorrhoeae and C. trachomatis from rectal swab samples have shown a higher burden of disease than would have been detected with traditional assays, including culture (1,6,(11)(12)(13)(14)(15).Despite a high prevalence of self-reported anal sex in the general population and some high-risk groups, there are no commercially available tests for chlamydia or gonorrhea that have regulatory approval for use with rectal swab samples (1,5,9,14). Problems of cross-reactivity with nongonococcal Neisseria species, which may frequently be found in the lower gastrointestinal tract, may have dissuaded commercial manufacturers from seeking regulatory approval for this sample type. Because of this, user verification is necessary to provide data to support their use. Few studies have evaluated the performance of NAATs for use with rectal samples; however, one such study tested self-collected rectal swabs with the Becton, Dickinson ProbeTec and Gen-...