Despite knowledge of the effects of toxic shock syndrome (TSS) toxin 1 (TSST-1) on the adaptive immune system, little is known about stimulation of the innate immune system, particularly epithelial cells. This study investigated the interactions of TSS Staphylococcus aureus and TSST-1 with human vaginal epithelial cells (HVECs) and porcine mucosal surfaces. When cocultured with HVECs for 6 h, TSS S. aureus MN8 proliferated, formed aggregates on the HVEC surfaces, and produced exotoxins. Receptor binding studies showed that 35S-TSST-1 bound to 5 × 104 receptors per HVEC, with saturation at 15 min. Affymetrix Human GeneChip U133A microarray analysis determined S. aureus MNSM (100 bacteria/HVEC) caused at least twofold up- or down-regulation of 410 HVEC genes by 6 h; these data were also confirmed with S. aureus MN8. TSST-1 (100 μg/ml) caused up- or down-regulation of 2,386 HVEC genes by 6 h. In response to S. aureus, the HVEC genes most up-regulated compared to those in controls were those coding for chemokines or cytokines-MIP-3α, 478-fold; GRO-α, 26-fold; GRO-β, 14-fold; and GRO-γ, 30-fold-suggesting activation of innate immunity. TSST-1 also caused up-regulation of chemokine/cytokine genes. Chemokine/cytokine gene up-regulation was confirmed by enzyme-linked immunosorbent assays measuring the corresponding proteins induced by S. aureus and TSST-1. S. aureus MN8, when incubated with porcine vaginal tissue, increased the flux of 35S-TSST-1 across the mucosal surface. This was accompanied by influx of lymphocytes into the upper layers of the tissue. These data suggest innate immune system activation through epithelial cells, reflected in chemokine/cytokine production and influx of lymphocytes, may cause changes in vaginal mucosa permeability, facilitating TSST-1 penetration.