Patients with toxic shock syndrome (TSS) had sequestered focal sites colonized or infected with Staphylococcus aureus significantly more frequently than did patients with scalded skin syndrome. Growth conditions within the infected foci of four patients with TSS included nearly physiological levels of pH, pO2, pCO2, protein, calcium, and magnesium. Two strains of S. aureus had typical TSS phenotypes (production of TSS toxin-1 [TSST-1] and protease), which were optimally expressed in vitro under conditions similar to those documented in vivo (6% CO2, pH 7.0, aerobic, high levels of protein). Altering any one of these growth factors significantly decreased production of TSST-1. In protein-containing media, depletion of divalent cations had a less-profound effect on organism growth and TSST-1 production. The unique in vivo environment in patients with non-menstrual TSS is similar to conditions associated with tampon use during menstruation. Growth conditions may play an important role in the expression of S. aureus phenotype and the pathogenesis of TSS.