The purification of staphylococcal beta-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic beta-hemolysin yielded one line of precipitate when tested against the original crude beta-hemolysin. The purified cationic beta-hemolysin was stable in the lyophilized state. Crude beta-hemolysin was dermonecrotic, whereas purified cationic beta-hemolysin was not dermonecrotic even after Mg(++) activation.