Fractionation of highly purified staphylococcal delta hemolysin by sucrose density gradient centrifugation, Sephadex G-150 and Bio-Gel A-5m gel filtration, carboxymethyl cellulose chromatography, and isoelectric focusing failed to separate the hemolytic, protoplast-lysing, and spheroplast-lysing activities of the preparation. The activities were reduced to a similar extent when delta hemolysin was treated with any of several phospholipids. The same was true when serum or proteolytic enzymes were used as inactivating agents. Cholesterol had no effect. The activities resisted heating at 80 C for 1 hr and were stable for at least 7 days at 5 C in 0.1 n sodium hydroxide, 0.1 n acetic acid, 6 m guanidine hydrochloride, 8 m urea, and 0.1% sodium lauryl sulfate.