Culture supernatants of 26 strains of Staphylococcus aureus possessing delta-hemolytic activity have been tested by immunodiffusion against a serum raised against purified delta-hemolysin from S. aureus CN 4108 (Newman D2). Supernatants from 14 strains of canine origin gave a reaction of partial identity with delta-hemolysin from CN 4108, whereas supernatants from all other strains had full identity. Delta hemolysin from one of these canine strains, CN 7450, was purified by ammonium sulfate fractionation and precipitation at pH 4.5. The physical, chemical, and biological properties of this toxin were compared with those of delta-hemolysin from CN 4108. Differences in molecular weight (as judged by Sepharose 6B chromatography), isoelectric point, and amino acid composition were found. Both toxins caused dermonecrosis in rabbits, lysed erythrocytes from several different species, and were inhibited by normal sera and phospholipids. Unlike delta-hemolysin from CN 4108, the hemolytic activity of delta-hemolysin from CN 7450 was found to be dependent on the incubation temperature over the range of 25 to 37 degrees C. Immunodiffusion results obtained with antisera raised against purified delta-hemolysin from CN 7450 indicated that delta-hemolysins from the canine strains were probably identical and confirmed that these differ immunologically from delta-hemolysin from CN 4108.