Retinoids function through conformational alterations of ligand-dependent nuclear transcription factors, the retinoic acid receptors, and retinoid X receptors. 9-cis-Retinoic acid is a known biological ligand for retinoid X receptors, but its synthesis pathway in vivo is largely unknown. Recently, we identified a cis-retinol dehydrogenase (cRDH) that oxidizes 9-cis-retinol to 9-cis-retinal. Since both the expression of cRDH mRNA and its substrate are found in liver, we studied 9-cis-retinol metabolism and 9-cis-retinoic acid biosynthesis in two hepatic-derived cell types, Hep G2 hepatoma cells and HSC-T6 stellate cells. Both cell lines accumulate similar amounts of 9-cis-retinol provided in the medium. However, Hep G2 cells preferentially incorporate all-trans-retinol when equimolar concentrations of all-trans- and 9-cis-retinol were provided. In contrast, HSC-T6 cells did not exhibit a preference between all-trans- and 9-cis-retinol under the same conditions. Esterification of 9-cis-retinol occurred in both cell types, likely by acyl-CoA:retinol acyltransferase and lecithin:retinol acyltransferase. In vitro enzyme assays demonstrated that both cell types can hydrolyze 9-cis-retinyl esters via retinyl ester hydrolase(s). In Hep G2 cells, 9-cis-retinoic acid synthesis was strongly inhibited by high concentrations of 9-cis-retinol, which may explain the low levels of 9-cis-retinol in liver of mice. Cell homogenates of Hep G2 can convert all-trans-retinol to 9-cis-retinal, suggesting that the free form of all-trans-retinol may be used as a source for 9-cis-retinol and, thus, 9-cis-retinoic acid synthesis. Our studies provide the basis for identification of additional pathways for the generation of 9-cis-retinoic acid in specialized tissues.