As reported previously squamous cell differentiation of rabbit tracheal epithelial (RTE) cells in culture is a multi-step process. This program of differentiation is inhibited by retinoic acid and retinol; retinoic acid is about 100 times more effective than retinol. To examine the metabolism of these agents in this in vitro model system, RTE cells were grown in the presence of all-trans-[3H]retinol or all-trans-3H]retinoic acid and their metabolites analyzed by high-pressure liquid chromatography. RTE cells converted most of the retinol to retinyl esters, predominantly retinyl palaaitate. A small fraction was metabolized to polar compounds, one of which coeluted with retinoic acid. After methylation this compound eluted as 13-cis-methyl retinoate and as all-trans-methyl retinoate. Conversion to 13-cis-retinol was also observed. All-trans-retinoic acid was rapidly taken up by RTE cells and converted to more polar (peak 1) and less polar (peak3) metabolites. A proportion of all-trans-[3H]retinoic acid was metabolized to 13-cis[3H]retinoie acid. These metabolic reactions appeared to be constitutive and were not induced by pretreatment with retinoic acid. The peak 1 metabolites were rapidly secreted into the medium whereas the peak 3 metabolites were retained by the cells and were not detected in the medium. Alkaline hydrolysis of the metabolites in peak 3 yielded retinoic acid, indicating the formation of retinoyl derivatives. Our results establish that RTE convert all-trans-retinol to 13-cis-retinol and retinoic acid. RTE can metabolize all-trans-retinoic acid to 13-cis-retinoic acid and to an unidentified ester of retinoic acid. © 1987.