In this study, for the first time, Clostridium histolyticum lethal factor was purified by ammonium sulfate precipitation of culture broth, centrifugation through an Amicon filter device and hydrophobic interaction chromatography. The purified lethal factor was devoid of proteolytic activity. At a concentration of 150 ng mL(-1) the lethal factor killed 50% of HeLa cells within 24 h of exposure. Abrogation of actin filaments, activation of caspases, fragmentation of nuclear DNA as well as ultrastructural changes indicated that the cell death occurred by apoptosis. The apoptotic action of the lethal factor is in agreement with changes induced in animal tissues by administration of C. histolyticum culture medium.