Keratins are a family of water-insoluble cytoskeletal proteins that form 10 nm desmosome-associated tonofilaments in almost all vertebrate epithelial cells. The subunit composition of keratin filaments is highly complex, with a total of 17 human epithelial keratins identified so far. Using monoclonal antibodies as a tool, we have recently demonstrated that all known human epithelial keratins can be divided into two subfamilies, A (for "acidic") and B (for "basic"), according to their charge properties and their immunoreactivities with AE1 and AE3 monoclonal anti-keratin antibodies. We have also demonstrated that, in the two subfamilies, keratins with the same "size-ranks" always follow similar rules of expression. Thus, the largest keratins of the two subfamilies, i.e., the 56.5K and 65-67K keratins of the A and B subfamilies, respectively, represent molecular markers for phenotypic keratinization, the second largest 55K (A) and 64K (B) keratins are unique to corneal epithelium, whereas the third largest 54'K (A) and 59K (B) keratins are characteristic of esophageal epithelium and other internal, nonkeratinized stratified epithelia. Based on these and other results, we have constructed a unifying model which can account for almost all known variations in keratin expression as a function of cell type, stages of embryonic development and differentiation, cellular growth environment, and diseases including neoplasms. To test the validity of this model and to provide a more detailed understanding of keratin structure and function, we will (i) determine whether keratins of the A and B subfamilies coexist in the same filaments at both the 10 nm tonofilament and 2 nm protofilament levels; (ii) characterize the epitopes defined by our monoclonal anti-keratin antibodies; (iii) define the relationship among the 56.5K keratin, 65-67K keratins, and filaggrin; (iv) isolate individual keratin species and study the structural and functional properties of keratin filaments reconstituted with various combinations of these purified keratins; and (v) localize specific keratins in hyperproliferative epidermis and in esophageal epithelium. Results from this study should lead to a better understanding of the functional significance of individual keratins and the mechanisms of normal and pathological epithelial differentiation.