The full-length cDNA of mouse K12 keratin was characterized by sequencing overlapping cDNA clones isolated from a mouse cornea cDNA library. Using Northern blot hybridization, the radio-labeled cDNA hybridized to a 1.9 kb mRNA from adult cornea, but not from other mouse tissues including snout, esophagus, tongue, and skin. During mouse development, corneas do not express K12 mRNA until 4 days postnatal when the epithelium begins to stratify as judged by Northern blot and in situ hybridization. In situ hybridization with 3H-labeled cDNA probe and immunohistochemical studies with antibodies against a synthetic oligo-peptide deduced from rabbit K12 cDNA demonstrate that this mouse K12 keratin is expressed in all cell layers of adult corneal epithelium, and the suprabasal layers, but not the basal layer of the limbal epithelium. Epidermal growth factor (EGF) has been shown to promote epithelium stratification of cultured chicken and human corneas in vitro. To examine whether EGF can promote K12 expression, EGF was administered to neonatal mice. The results indicate that EGF retards K12 expression by corneal epithelial cells, even though it promotes corneal epithelial stratification during mouse development. Taken together, our results demonstrate that the expression of K12 keratin is cornea-specific, differentiation-dependent, and developmentally regulated.