Alpha haemolysin, produced by Escherichia coli, grown in a chemically defined medium, was purified 19-fold and the endotoxin content reduced 2176-fold by ultrafiltration and glycerol-gradient ultracentrifugation. Immunodiffusion of purified alpha haemolysin (PH) against antiserum to crude haemolysin (CH) revealed only one precipitation line. PH was cytotoxic in nanogram amounts for mouse-fibroblast 3T3 cells, and the cytotoxicity exhibited proportional dose-response and time-course kinetics. The cytotoxic and haemolytic activities of PH were neutralised by immunoglobulins to CH. A mutant, produced by treating the haemolytic wild type with mitomycin C, possessed all of the biochemical characteristics of the wild type with the exception that the extracellular products of the mutant were non-haemolytic and non-cytotoxic.