Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal a-toxin indicated the presence of a lytic factor other than a-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified a-toxin (HP a-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure a 12S-toxin. The interaction of HP a-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The a 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An a 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the a 12S molecule. Lipid monolayer experiments showed that HP a-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP a-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to a-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to a 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.