CCAAT-enhancer binding proteins (C/EBP) are basic region/leucine zipper (bZIP) transcription factors selectively expressed during the differentiation of liver, adipose tissue, blood cells, and the endocrine pancreas. Here we show that C/EBP isoforms are differentially expressed in the skin. BALB/MK keratinocytes incubated in 0.12 mM calcium medium undergo a differentiation program featuring growth-arrest at 24-48 h, keratin K10 gene expression beginning at 24 h, and apoptosis commencing at 48 h. Within this framework, western immunoblot analysis and immunohistochemistry reveal that C/EBP alpha increases 5-fold at 1-2 d and remains elevated, C/EBP beta rises 2-fold at 2-4 d and gradually falls, and CHOP rises 9-fold in the first 24 h then returns rapidly to baseline. Several products of alternative translation are observed in BALB/MK cells, i.e., 42 kDa and 30 kDa forms of C/EBP alpha, and 32 kDa and 20 kDa forms of C/EBP beta. By immunohistologic examination of human, rat, and mouse skin, all three transcription factors are highly expressed within epithelial compartments in a spatially restricted distribution. C/EBP alpha is concentrated in the upper epidermis in a predominantly cytoplasmic location within cells, whereas the highest levels of C/EBP beta and CHOP are seen in the mid-epidermis, mainly within nuclei. High levels of C/EBP beta and CHOP (but not C/EBP alpha) are also observed in hair follicles and sebaceous glands. The identity of these factors in the epidermis is confirmed by western immunoblot analyses. In summary, C/EBP are expressed in a differentiation-associated manner in the skin, and may play an important role in regulating one or more aspects of the epidermal differentiation program.