We have used antibody to chicken gizzard alpha-actinin to identify and localize this molecule in chicken intestinal epithelium. The antibody binds only to alpa-actinin when tested against a crude extract of chicken gizzard. Extracts of purified epithelial cells contain a molecule which has a subunit molecular weight of 100,000 on sodium dodecyl sulphate gels and which is able to inhibit the interaction of alpha-actinin antibody and 125I-labeled chicken gizzard alpha-actinin. By indirect immunofluorescence, alpha-actinin is localized in the apical portion of chicken intestinal epithelial cells. Ethanol-fixed cryostat sections of intestine taken through the apical portion of the epithelial cells and in a plane perpendicular to the long axis of the cells show that alpha-actinin is organized in a polygonal pattern which corresponds to the outlines of the polygonally packed epithelial cells.