Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2-II, respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2-I, respectively) with ADP-ribosyltransferase activity that depolymerizes the actin cytoskeleton. After internalization, it was found that C2 and Iota toxins were not routed to the Golgi apparatus and exhibited differential sensitivity to inhibitors of endosome acidification. While the C2-I component of C2 toxin was translocated into the cytosol from early endosomes, translocation of the Ia component of Iota toxin occurred between early and late endosomes, was dependent on more acidic conditions, and uniquely required a membrane potential gradient.