The hemolytic activity of toxic shock syndrome isolates of Staphylococcus aureus is enhanced when agarose is substituted for agar in blood plates or when strains are grown in liquid culture in the presence of 20% (vol/vol) CO2 in air. Hemolytic activity of a representative panel of toxic shock syndrome isolates was rigorously assessed both on blood agar and in liquid culture to unequivocally identify the predominant hemolysins produced. As determined by isoelectric focusing and Western immunoblotting, 15 of 15 TSS isolates produced gamma-lysin and 10 of 15 produced delta-lysin. None produced beta-lysin, and only 2 of 15 produced alpha-lysin. The low rate of alpha-lysin production was a most striking characteristic, since all strains were found to have the alpha-lysin gene by Southern blot hybridization.