The hairless (hr) gene is a putative transcriptional factor whose mutations lead to hair loss in animals and humans. As a step toward understanding the role of the hr gene, we investigated the expression of hr mRNA in mouse keratinocyte differentiation. Treatment of mouse primary keratinocyte cultures with phorbol-12-myristate-13-acetate (PMA) reduced DNA synthesis and sequentially induced an up-regulation of p21Cip1/WAF1 (p21), hr and involucrin (inv) mRNAs in a time-dependent fashion, suggesting that an increase in hr gene expression is associated with keratinocyte differentiation. This up-regulation was blocked by the RNA synthesis inhibitor actinomycin D. However, an increase in hr mRNA, but not in inv mRNA, was seen in cells treated with the protein synthesis inhibitor cycloheximide, suggesting that new protein synthesis is involved in the suppression of hr transcription or in the degradation of hr mRNA in the steady state. The up-regulation of hr mRNA expression by PMA was blocked by the protein kinase C (PKC) inhibitor, GF109203X. These data indicate that PKC activation is involved in the up-regulation of hr mRNA expression during mouse keratinocyte differentiation.