Large amounts of an extremely potent extracellular cytolysin produced by the halophilic bacterium Vibrio damsela were obtained free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-100, and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The cytolysin is heat labile and protease sensitive and has a molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of ca. 69,000 and an isoelectric point of ca. 5.6. The first 10 amino-terminal amino acid residues of the cytolysin are Phe-Thr-Gln-Trp-Gly-Gly-Ser-Gly-Leu-Thr. The cytolysin was very active against erythrocytes from 4 of the 18 animal species examined (mice, rats, rabbits, damselfish) and against Chinese hamster ovary cells and was lethal for mice (ca. 1 microgram/kg, intraperitoneal median lethal dose). Lysis of mouse erythrocytes by the cytolysin is a multi-hit, at least two-step process consisting of a temperature-independent, toxin-binding step followed by a temperature-dependent, membrane-perturbation step(s).