Staphylococcal beta hemolysin from the 681 strain of Staphylococcus aureus grown in a Heart Infusion dialysate semisolid medium under 10% carbon dioxide was obtained in an immunoelectrophoretically pure form by a combination of procedures of precipitation with 2 volumes of acetone followed by chromatography on diethylaminoethyl cellulose at pH 6.0. The acetone precipitation procedure did not show any deleterious effect on the hemolytic activity of the beta hemolysin unless the precipitate was left in contact with the acetone for at least 4 hr. The crude preparations contained two types of beta hemolysin. One of these represented the major portion of the total activity of beta hemolysin and behaved as a cation. The other represented a minor (1/5,000) portion of the total beta hemolysin activity and behaved as an anion. These active principles were designated as cationic and anionic beta hemolysins, respectively. An unexpected increase in the total beta hemolysin activity of the crude preparations was noted when these were concentrated by dialysis against polyethylene glycol (20 m). This effect was probably due to polyethylene glycol. A further unexpected increase in the titer of the acetone-precipitated preparations occurred when these were lyophilized. The reason for this incremental increase is not known. It may be due to fragmentation of the beta hemolysin.