C2 toxin elaborated by Clostridium botulinum types C and D is composed of two nonlinked protein components and has enterotoxic activity, for which the cooperation of these two components is necessary. In the present study, the binding of components I and II, the two components of C2 toxin, to isolated epithelial cells and brush borders of mouse intestine was examined. Immunofluorescence studies showed that component II, either trypsinized (T-II) or untrypsinized (UT-II), bound to the cells and the brush borders of mouse intestine, whereas component I alone did not. The binding of I was observed only when the cells and the brush borders were reacted with T-II, but not when they were reacted with UT-II. These results are consistent with the fact that the biological activities of C2 toxin are elicited by the combination of I and T-II, but not of I and UT-II. The in vitro binding of I and II to isolated brush borders of mouse intestinal cells also showed similar binding characteristics. The binding of I and II to brush borders was rapid and not temperature dependent. Ultracentrifugal analysis revealed that both I and T-II bound to microvillous membranes of the intestinal cells. The data from the present study indicate that the enterotoxic activity of C2 toxin is initiated by the binding of T-II to the microvillous membrane of intestinal cells followed by that of I, for which the site of the cell membrane is induced by the binding of T-II, but not of UT-II.