We have localized capping protein in epithelial cells of several chicken tissues using affinitypurified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by con-focal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.