Nasal carriage of Staphylococcus aureus is often a prelude to infection with the same strain. The prevailing assumption has been that colonized individuals carry a single strain. The present study investigated the frequency of simultaneous nasal carriage of multiple strains of S. aureus. Three bacterial colonies from plated samples from colonized subjects were initially compared by pulsed-field gel electrophoresis. Fourteen of 148 S. aureus-positive samples demonstrated at least a difference of a single band; 7 of these 14 samples contained different strains, and 3 of these 7 also belonged to different accessory gene regulator (agr) types. The remaining 7 samples contained clonally related isolates; 3 of these 7 contained pairs that differed by the presence or absence of the staphylococcal chromosomal cassette mec type IV. A mathematical model that we developed predicted that approximately 6.6% of S. aureus-colonized individuals carry >1 strain. The present study demonstrates that carriage of discordant S. aureus strains in individuals with nasal colonization occurs regularly and suggests that the nares are likely sites for horizontal genetic exchange among strains.