The ultrastructural localization of three cytoskeletal proteins, alpha-actinin, tropomyosin, and vinculin, in the brush border of epithelial cells of chicken small intestine and the smooth muscle cells of chicken gizzard was studied by immunofluorescence and immunonelectron microscope labeling of frozen sections of lightly fixed, intact tissues. In the immunoelectron microscope studies, a recently described new type of electron-dense antibody conjugate, imposil-antibody, has been successfully used, along with ferritin-antibody conjugates, in single and double immunolabeling experiments. In the intestinal brush border shows that vinvulin is sharply confined to the junctional complex close to the membrane region of the zonula adherens, in distinct contrast to the more diffuse distributions of the other two proteins. In the smooth muscle cells, the labeling patterns show that vinculin is sharply confined to the membrane-associated dense plaques, closer to the membrane than the alpha-Actinin is also present in the cytoplastic dense bodies, from which vinculin is absent. Tropomyosin is present diffusely distributed in the cytoplasm, but absent from both dense plaques and dense bodies. These findings with the muscle cells demonstrate, therefore, that the dense plaques and dense bodies are chemically and structurally distinct entities. The results with both tissues, along with those in previous papers (Geiger, 1979, Cell. 18:193-205.; Geiger et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:4127-4131), suggest that vinculin may play an important and widespread role in the linkage of actin-containing microfilament bundles to membranes.