Staphylococcal pyrogenic exotoxin type C was purified by differential precipitation with ethanol and resolubilization in water followed by thin-layer isoelectric focusing. The purified toxin migrated as a homogeneous protein when re-electrofocused in polyacrylamide (isoelectric point, 7.2), when subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (molecular weight, 22,000 daltons), and when reacted against hyperimmune antisera in an Ouchterlony immunodiffusion plate. A 2200-fold purification of the toxin could be obtained. Pyrogenic exotoxin C was elaborated by all of 44 Staphylococcus aureus isolates from patients with toxic shock syndrome when the strains were tested in a blind manner, but five of 37 control isolates produced toxin (p less than 0.001). Furthermore, all of 91 additional isolates from patients with toxic shock syndrome from diverse geographic locations were positive for toxin. In contrast, 158 of 609 (26%) S. aureus isolates not associated with toxic shock syndrome and one of 22 Staphylococcus epidermidis isolates were positive for pyrogenic exotoxin type C. The biological properties of pyrogenic exotoxin C included pyrogenicity, enhancement of susceptibility to endotoxin shock, enhancement of skin reactivity to give a positive Dick reaction, suppressions of IgM synthesis, and nonspecific T-lymphocyte mitogenicity.