We compared ligase chain reaction (LCR) assay with standard culture for the detection of pharyngeal Neisseria gonorrhoeae infection in men who have sex with men (MSM) presenting at a sexually transmitted diseases clinic in San Francisco. Pharyngeal specimens were obtained from 200 MSM who reported performing fellatio during the previous 2 weeks. Confirmatory testing of discrepant specimens was conducted using N. gonorrhoeae pilin proteins. Prevalence of pharyngeal N. gonorrhoeae was 6% by culture or 11% by LCR. The sensitivity and specificity of LCR were 94.7% and 97.8%, respectively, compared with values of 47.4% and 100% for culture. Prevalence of pharyngeal N. gonorrhoeae infection, as determined by DNA amplification testing, was higher than that suggested by traditional culture. Results support the use of DNA amplification testing in the oropharynx. The high prevalence of pharyngeal N. gonorrhoeae infection among MSM suggests that routine screening should be considered in efforts to reduce the burden of gonorrhea in this population.