Villin, a 95,000 dalton polypeptide of intestinal brush border which is known to bundle or sever actin filaments in a Ca++-dependent manner, was localized in rat and chicken intestinal epithelium by means of immunocytochemistry at the light- and electron-microscopic levels. Specific antibodies to villin were raised in rabbits immunized with villin purified from chicken intestinal epithelium. Anti-villin bound selectively to the microvillus filament bundle from its tip down to the rootlets. These findings indicate that the well-known stability of rootlet filaments towards elevated Ca++ ion concentrations cannot be explained by the absence of villin. Therefore additional factors must exist which prevent the rootlets from Ca++-villin mediated disassembly.