Villin, a 95,000-dalton protein, is a major component of microvillus cores isolated from intestinal brush borders. In this study, we compared the Ca2+-dependent action of this protein on actin filaments with that of cytochalasin B, a fungal metabolite that binds to the "barbed" end of actin filaments and nuclei. We found that substoichiometric levels of villin inhibit actin filament elongation and self-association in a cytochalasin-like manner. In addition, the protein releases membrane-bound F-actin in the absence of high shear force, probably by severing the filaments. The filament fragments formed in the presence of villin, as well as a nucleating complex consisting of villin and actin, bind stoichiometric amounts of [3H]cytochalasin B with high affinity. The results of this study indicate that both villin and cytochalasin B bind to the same end of actin filaments, yet differ in their binding sites.