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/EBPα, C/EBPβ, and C/EBPδ 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/EBPα protein and 35 kDa C/EBPβ protein. Immunohistochemical localization of C/EBPα and C/EBPβ in intact interfollicular epidermis revealed that C/EBPβ 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/EBPα 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/EBPα and C/EBPβ was greatly diminished as both the intensity of nuclear staining and the number of cells expressing C/EBPα and C/EBPβ were reduced. In isolated primary mouse keratinocytes, calcium-induced differentiation was accompanied by specific temporal changes in the expression of C/EBPα, C/EBPβ, and C/EBPδ mRNA and C/EBPα and C/EBPβ 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.