A hemolysin, botulinolysin, produced by Clostridium botulinum was purified to homogeneity and characterized. First, a strain of C. botulinum type C, strain C-203 Tox, which produced a large amount of hemolysin, was selected, and optimal culture medium and conditions for its production of hemolysin were determined. The hemolysin produced in the culture supernatant of this strain under optimal conditions was purified by a combination of ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, Sephadex G-75 gel permeation chromatography, and SP-Toyopearl 650 M cation-exchange column chromatography, with a recovery of 12%. The purified hemolysin gave a single protein band in polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulfate (SDS). The protein in this band in PAGE with SDS was estimated to have a molecular weight of 58,000 and was immunostained with a neutralizing monoclonal antibody. In PAGE without SDS, the hemolytic activity corresponded in position to the single protein band. The pI of the hemolysin was 8.4. Amino acid analysis of the purified hemolysin indicated the presence of four half-cystine residues per molecule. The purified hemolysin had a specific activity of 2,100 hemolytic units per microgram of protein on rabbit erythrocytes. It was activated by SH compounds, inhibited by cholesterol, and heat labile. The optimum pH for hemolysis was 6.0 to 7.0. Rabbit, human, and guinea pig erythrocytes were the most susceptible to the hemolysin, while sheep, mouse, rat, and chicken erythrocytes were much less susceptible. The purified hemolysin had a lethal effect in mice and was cytotoxic for some cultured cells: its 50% lethal dose in mice was 310 ng, and its 50% cytotoxic dose for Vero cells was 120 ng/ml.