Lipid bilayer experiments were performed with the C2-II binding component of the ADP-ribosylating C2 toxin from Clostridium botulinum. The trypsin-activated but not the nonactivated form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 55 pS in 0.1 M KCl and were found to be cation-selective and voltage-dependent. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. Incubation of the activated C2-II binding component with antibodies against C2-II or with C2-I toxin inhibited channel formation to a large extent. Addition of chloroquine, a known inhibitor of endocytosis in cells, led to a dose-dependent decrease of the C2-II-induced membrane conductance. This result suggested that the activated C2-II component contains a binding site for chloroquine inside the channel. It is discussed that the channels formed by C2-II component are involved in the translocation of C2-I toxin across the target cell membrane.