Rabbit tracheal epithelial cells grown on fibronectin-albumin-vitrogen coated dishes in serum-free and vitamin A-free medium undergo terminal squamous differentiation under a variety of conditions. This differentiation occurs in several stages. First cells undergo terminal cell division; cells accumulate in G0/G1 phase of the cell cycle and loose the ability to form colonies. This terminal cell division occurs at high density or is induced at low density when epidermal growth factor is omitted from the medium or when TGFBeta is added. This is followed by the expression of a squamous phenotype that is characterized by the induction of a squamous morphology, increase in transglutaminase and cholesterol sulfate levels, increase in the expression of a 48 and 56 kd keratin and the formation of cross-linked envelopes. The increase in cholesterol sulfate levels appears to be related to an enhancement in sulfotransferase activity. High calcium concentration promotes the expression of the squamous phenotype. Retinoic acid has no effect on the commitment to terminal cell division but inhibits the expression of the squamous phenotype; retinoic acid inhibits the increase in transglutaminase and cholesterol sulfate levels the alterations in keratin expression and the formation of cross-linked envelopes. To study the regulation of differentiation at the molecular level, we established a cDNA library using poly (A)+ RNA from squamous differentiated cells and isolated recombinant cDNA clones that hybridize with mRNA's isolated from squamous differentiated cells but not from undifferentiated and retinoic acid-treated cells. This indicates that these clones represent gene products that function as markers for differentiation. We are in the process to analyze at what level these gene products are regulated and how retinoids control them.