Immunofluorescence and microinjection experiments have shown that vinculin (molecular weight 130,000) is localized at adhesion plaques of fibroblasts spread on a solid substrate. We found that this protein affects actin filament assembly and interactions in vitro at substoichiometric levels. Vinculin inhibits the rate of actin polymerization under conditions that limit nuclei formation, indicating an effect on the filament elongation step of the reaction. Vinculin also reduces actin filament--filament interactions measured with a low-shear viscometer. Scatchard plot analysis of the binding of 3H-labeled vinculin to actin filaments showed that there is one high-affinity binding site (dissociation constant=20 nM) for every 1,500-2,000 actin monomers. These results suggested that vinculin interacts with a specific site located at the growing ends of actin filaments in a cytochalasin-like manner, a property consistent with its proposed function as a linkage protein between filaments and the plasma membranes.