Outbreaks of skin and soft tissue infections mediated by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are being reported with increasing frequency among men who have sex with men (MSM). However, the potential role of asymptomatic colonization with this organism in perpetuating these infections is unclear. The purpose of this cross-sectional study was to determine the prevalence of colonization with CA-MRSA among a cohort of 500 MSM recruited from two inner city clinics in Toronto, Canada. Following the provision of informed consent, subjects completed a questionnaire capturing demographic and clinical variables, which may be associated with MRSA colonization. A nasal swab for MRSA was collected from each subject, and instructions were provided regarding the self-collection of a rectal swab. Cultured MRSA underwent pulsed-field gel electrophoresis and virulence testing for Panton-Valentine leukocidin gene expression. The prevalence of CA-MRSA colonization was 1.6% (95% CI: 0.5-2.6%).