The relationship among tuft protein derived from mature human molars, human skin keratins, and the developing enamel matrix of the rat incisor was investigated using polyclonal antibodies in immunocytochemical and Western blotting techniques. Antibodies to tuft protein and keratin cross-reacted with proteins in the Mr range 50–70 K from demineralized developing enamel, enamel organ, human skin, and tuft extract. An immunocytochemical technique was used to locate the site of cross-reactivityin situ within secretory ameloblasts, enamel, and keratinized epithelium at the ultrastructural level. Antibodies to keratin cross-reacted with cytoplasmic tonofilaments and those inserted into desmosomes. Antibodies to tuft protein, however, did not cross-react with cytoskeletal components but produced labeling of the golgi and secretory vesicles. Labeling with this antibody was also observed within the stratum granulosum of the rat foot pad. It is concluded that tuft protein contains secretory products of the ameloblast that may represent a less specialized product of other epithelial tissues, perhaps related to the keratins.