The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that undergo sequential changes in gene expression during differentiation. CCAAT/enhancer binding proteins (C/EBP) are members of the bZIP family of DNA binding proteins/transcription factors. Northern analysis demonstrated that C/EBPalpha, C/EBPbeta, and C/EBPdelta mRNA are expressed in mouse epidermis and their mRNA levels were generally greater than those observed in other tissues known to express high levels of C/EBP. Western analysis of isolated epidermal cell nuclei demonstrated the presence of a 42 and 30 kDa C/EBPalpha protein and 35 kDa C/EBPbeta protein. Immunohistochemical localization of C/EBPalpha and C/EBPbeta in intact interfollicular epidermis revealed that C/EBPbeta expression is exclusive to the nuclei of a three-cell cluster of suprabasal keratinocytes that is morphologically consistent with the central column of the epidermal proliferative unit, and that C/EBPalpha is expressed in the nuclei and cytoplasm of suprabasal keratinocytes and weakly expressed in a perinuclear manner in some basal keratinocytes. In squamous cell carcinomas the expression of C/EBPalpha and C/EBPbeta was greatly diminished as both the intensity of nuclear staining and the number of cells expressing C/EBPalpha and C/EBPbeta were reduced. In isolated primary mouse keratinocytes, calcium-induced differentiation was accompanied by specific temporal changes in the expression of C/EBPalpha, C/EBPbeta, and C/EBPdelta mRNA and C/EBPalpha and C/EBPbeta protein. These results implicate a role for the C/EBP family in the regulation of genes involved in or specifically expressed during the process of squamous differentiation in epidermis.