Proteins of the ezrin-radixin-moesin family are ubiquitous constituents of the submembrane cortex, especially in epithelial cells. Earlier biochemical results suggested that a protein of this family occurs in the hair bundle, the cluster of actin-filled stereocilia that serves as the mechanoreceptive organelle of each hair cell in the inner ear. We prepared antipeptide antisera directed against chicken radixin and ezrin and demonstrated their specificity and absence of crossreactivity. When used in immunocytochemical studies of isolated hair cells, anti-radixin produced an intense band of labeling at the bases of hair bundles from the chicken, frog, mouse, and zebrafish. Electron microscopic immunocytochemistry disclosed that radixin labeling commenced in the stereociliary taper, peaked in the lower stereociliary shaft, and declined progressively toward the hair bundle's top. Labeling with anti-ezrin produced no signal in hair bundles. Radixin is thus a prominent constituent of stereocilia, where it may participate in anchoring the "pointed" ends of actin filaments to the membrane.