Biochemical analyses have previously shown that palmar and plantar epidermis, unlike the epidermis of other body sites, contain cytokeratin 9 (Mr 64,000), an unusually large acidic (type I) cytokeratin. Guinea-pig antibodies that specifically and selectively react with bovine and human cytokeratin 9 were used for the immunocytochemical identification of cytokeratin 9 in adult and fetal human epidermis from various body sites. In the epidermis of palms and soles, antibodies against cytokeratin 9 stained a high proportion of the keratinocytes in suprabasal locations. These suprabasal cytokeratin-9-positive keratinocytes were often arranged in vertical columns and concentrated around intraepidermal sweat-gland ducts, but they sometimes also formed extended continuous sheets. In contrast, another type-I component, cytokeratin(s) 10/11, was uniformly distributed among suprabasal keratinocytes. By double-labeling immunofluorescence microscopy using a monoclonal antibody against cytokeratin(s) 10/11, we found that cytokeratin 9 usually appears in cells located one or two layers above the cells in which cytokeratin(s) 10/11 is detected, indicating that most keratinocytes expressing cytokeratin 9 also express cytokeratin(s) 10/11. At other body sites, cytokeratin 9 was only detected in sparsely distributed keratinocytes localized in upper epidermal layers; these cells were scattered or formed small clusters, and often exhibited a conspicuous association with the epidermal portion of eccrine sweat-gland ducts. During human fetal development, cytokeratin 9 was first detected at week 15 of gestation in some suprabasal cells of the foot-sole epidermis and, occasionally, in basal cells. At later fetal stages, most of the cytokeratin-9-positive cells appeared in clusters that were mainly concentrated in glandular ridges and interridges. Our results show that two major types of terminally differentiating keratinocytes can be distinguished in human epidermis, i.e. those that do and those that do not express cytokeratin 9. This special program of keratinocyte differentiation identified by the presence of cytokeratin 9 appears to be related to the morphogenesis of palm and sole epidermis, where this protein is expressed early in fetal life. Possible biological functions of this subset of cytokeratin-9-positive keratinocytes are discussed.