A comprehensive analysis of Staphylococcus aureus superantigen (SAG) genes was undertaken in isolates from a major hospital and compared with isolates from patients with toxic shock syndrome (TSS). Polymerase chain reaction (PCR) analysis included recently discovered SAGs. Staphylococcal enterotoxin (SE) G and SEI were uniquely expressed in genital isolates. Genital isolates were similar to TSS isolates, although the latter frequently expressed TSS toxin 1. Both had a high frequency of SEG/SEI and a high number of SAG genes per bacterium. Detection of an SAG gene by PCR correlated with positive results in functional assays for SAG activity. Levels of serum antibodies to SEG and SEI, but not to other superantigens, were higher in healthy women than in men and served as an independent measure of the higher frequency of exposure to SEG/SEI among women. Together, the data suggest a role for SEG/SEI or closely linked genes in the adaptation of S. aureus to the genital mucosa environment.