A multilayered, continuously proliferating keratinocyte cell culture has been produced from rat sublingual epithelium. The rate of growth of the cultures was stable throughout long-term culture. Retinoic acid (3.3 microM) inhibited the keratinization of these cultures. Morphological changes included total loss of tonofilaments within 7 days, decrease in desmosomes, an increase in intercellular spaces, absence of thickened plasma membranes, and elongated and more numerous cytoplasmic projections. Exposure to retinoic acid (3.3 microM) for 33 days did not affect the growth rates of the cultures, as estimated from the protein and DNA content per flask. Retinoic acid (3.3 microM) reduced the polyacrylamide gel electrophoresis protein profile within 3 days of treatment and produced reductions in the incorporation of amino acids into keratins of molecular weights 62,000 and 60,000 within 24 h. All five keratin polypeptides showed a reduced incorporation rate after treatment for 3 days. This inhibition was reversible. Protein synthesis of nonkeratins was not detectably affected by retinoid treatment.