A basic protein, solubilized in buffered salt solutions from keratohyalin granules of newborn rat epidermis, has been purified by ion-exchange chromatography. The relative molecular weight of the protein was determined as 12 800 +/- 200 from its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is relatively rich in lysine, glycine, alanine, and valine which together comprise about 60% of the total amino acid residues. Using an antibody to this protein, which we have designated fraction 4, we have found that it is specific to rat epidermis and is not present in any other rat tissues or in epidermal extracts from other species. The cells of the four epidermal layers were separated and the amount of fraction 4 in each cell layer was measured by radioimmunoassay. The protein is localized mainly in the upper layers of epidermis. The protein, which binds to DNA, appears in the epidermis just prior to birth, increases during the first week of post-natal life and declines sharply thereafter. Fraction 4 represents about 7% of the total solubilized protein in 7-day-old rat epidermis.