Sterile, cell-free filtrates of five strains of coagulase-positive phage group II staphylococci that were cultured in vivo produced exfoliation in newborn mice after intraperitoneal, oral, or subcutaneous administration. Filtrates prepared in a similar manner from coagulase-positive staphylococci of other phage groups failed to produce exfoliation. All filtrates contained staphylococcal alpha hemolysin, the activity of which could be neutralized with specific antiserum. The toxin responsible for the exfoliative phenomenon was a macromolecular ampholyte with a molecular weight of the same order as that of alpha hemolysin. Its behavior was similar to that of alpha hemolysis on gel filtration on Sephadex, Pevikon-block electrophoresis, ultrafiltration, and ultracentrifugation. It was partially separated from alpha hemolysin by precipitation with ammonium sulfate. Isoelectric focusing showed that the exfoliative toxin had an isoelectric point of 7.0 and could be completely separated from the more basic alpha hemolysin by this technique. After purification, approximately O.5 µg of the powdered toxin was sufficient to cause exfoliation in the newborn mouse. The identification and separation of the staphylococcal exfoliative toxin and the production of a skin lesion with a partially purified preparation establish this toxin as the etiologic agent of the skin changes seen in children with the staphylococcal scalded-skin syndrome.