The four major epidermal keratins (65-67K, 58K, 56K, and 50K) have been localized in various cell layers of normal human epidermis. Guinea pig antisera and mouse monoclonal antibodies were prepared against human epidermal keratins and were characterized with respect to their specificity to individual keratin polypeptides by the immunoblot technique. These antibodies were used to stain vertical frozen sections of skin, and to identify keratins extracted from serial, horizontal skin sections. The results indicate that: (1) a 65-67K keratin component is limited to the suprabasal layers, (2) a 58K keratin is present throughout the epidermis, (3) a 56K keratin appears to be made only in cells above the basal layer, possibly in the upper spinous or granular layer, and (4) a 50K keratin is present in all living layers but is largely eliminated during stratum corneum formation. The 65-67K and 56K keratins, which are characteristic of suprabasal, terminally differentiated keratinocytes, may be regarded as molecular markers of keratinization.