The effect of separate injections of two components of Clostridium perfringens iota toxin, designated Ia and Ib components, on the biological activities of the toxin was investigated. The intravenous injection of one component within 120 min after the injection of the other component killed mice. The activity of iota toxin was abolished by anti-Ia or anti-Ib antiserum. On the other hand, when Ib component was intravenously administered to mice given anti-Ia antiserum within 120 min after the intravenous injection of Ia component, the lethal activity was completely neutralized, but when Ia component was injected into mice that were given anti-Ib antiserum over 5 min after the injection of Ib component, the activity was not neutralized. The separate injections of Ia and Ib components in skin of guinea pigs indicated dermonecrosis at the injection site of Ib components, but not at the site of Ia components. Furthermore, when one component was intradermally injected in guinea pigs and then the other intraperitoneally, the dermonecrotic activity of the toxin was observed at the intradermal injection site of Ib component, but not at that of Ia component. From the data, it appears that the lethal and dermonecrotic activities of iota toxin are initiated by the binding of Ib component to specific sites on tissues.