Self-collected glans and rectal swab specimens from men who have sex with men (MSM) may be appropriate, convenient specimens for testing. We evaluated the use of self-collected swabs for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by a transcription-mediated amplification test (AC2; Aptima Combo 2; Gen-Probe Inc.) and a strand displacement amplification test (SDA; ProbeTec; Becton Dickinson Co.) in MSM seen at the city sexually transmitted disease clinic in San Francisco, CA. For the glans swab specimen, subjects enrolled early in the study rolled a Dacron swab across the meatus three times (method 1). A slightly more invasive procedure was performed later in the study: the subjects inserted the swab 1/4 in. into the urethra, rotated the swab, and then withdrew the swab (method 2). MSM self-collected a rectal swab specimen and also provided first-catch urine (FCU). Additional rectal swab samples were then obtained by the clinician. For the detection of C. trachomatis and N. gonorrhoeae, all swabs were evaluated by AC2 and SDA, FCU was tested by AC2, and the clinician-collected rectal swabs were cultured. A rectal true-positive (TP) result was defined as a culture-positive result for C. trachomatis or N. gonorrhoeae, two or more positive nucleic acid amplification test (NAAT) results, or a single NAAT-positive result confirmed by an alternate amplification method (the Aptima C. trachomatis or N. gonorrhoeae test). A glans TP result was defined as a positive result for FCU, positive results for both glans specimens (one tested by AC2 and one tested by SDA), or a positive result for a single glans specimen confirmed by an alternate amplification method. The prevalence rates of C. trachomatis and N. gonorrhoeae by testing of FCU were 6.8% (60/882 specimens) and 12.2% (108/882 specimens), respectively. Mixed results were obtained with the glans swab: N. gonorrhoeae detection by AC2 and SDA (method 1) had the best performance (sensitivities, >92%) with samples from a population with a higher prevalence of infection, but their performance for the detection of C. trachomatis was poor and varied by collection method (sensitivities, 56 to 68%). The prevalence rates of C. trachomatis and N. gonorrhoeae in the rectum were 7.3% (66/907 specimens) and 9.4% (83/882 specimens), respectively. The sensitivities of the tests with self-collected and clinician-collected rectal swab specimens were comparable (for C. trachomatis, 41% and 44%, respectively, by SDA and 82% and 71%, respectively, by AC2; for N. gonorrhoeae, 77% and 68%, respectively, by SDA and 84% and 78%, respectively, by AC2). AC2 and SDA were far superior to culture for the detection of C. trachomatis and N. gonorrhoeae in the rectum, with both tests detecting at least twice as many infections. While we found self-collected rectal swabs from MSM to be valid specimens for testing, the sensitivities of the tests with glans swab specimens were disappointing except for those from patients with symptomatic N. gonorrhoeae infections. Self-collected glans swab specimens may not be appropriate for the detection of C. trachomatis or for the detection of N. gonorrhoeae in low-risk or asymptomatic patients by AC2 and SDA, and we would not recommend their use on the basis of our results. Further studies are needed.