Cornifins/small proline-rich proteins (SPRRs) belong to a family of proline-rich proteins that function as cornified envelope precursors. We report here an immunohistochemical analysis of human cornifin-alpha and -beta expression in several stratified squamous epithelia. In normal human skin, cornifin-alpha was expressed in the granular layer of the epidermis of palmoplantar skin, in the inner lining cells of the follicular infundibulum, and in the inner root sheath of the hair follicle. It was also expressed in the upper squamous layers of the oral, esophageal, and vaginal epithelia. Cornifin-beta was detected in oral, esophageal, and vaginal epithelia, but not in normal skin. Immunoblot analysis revealed quantitative differences in cornifin-alpha expression in skin from different regions. Studies of specimens from various skin diseases showed that (i) cornifin-alpha was upregulated in inflammatory skin diseases, hyperplastic lesions, and in well-differentiated squamous cell carcinomas (SCCs), (ii) the expression of cornifin-beta was absent in inflammatory skin but was detected in highly differentiated keratinocytes in well-differentiated SCCs of the skin and some other hyperproliferative skin lesions, and in SCCs of the oral mucosa and esophagus. Northern blot analysis revealed that cornifin-alpha mRNA was present in all the squamous epithelial tissues studied, whereas cornifin-beta mRNA was expressed in oral mucosal epithelia and verrucous carcinoma of the skin but neither in normal nor in psoriatic skin. These results indicate that (i) the amount of cornifin alpha/SPRR1 expression in normal human skin depends on the body region, (ii) cornifin-alpha/SPRR1, but not cornifin-beta, contributes to the integrity of the hair follicle, and (iii) the expression of cornifin-beta is induced in some hyperplastic skin diseases only when the keratinocytes undergo extensive squamous differentiation.