Oxidation of retinol to retinaldehyde is an important first step in the biosynthesis of a biologically active retinoic acid, which serves as an activating ligand for retinoic acid receptors. Retinol can be oxidized by a variety of different oxidoreductases found in both the cytosolic and the microsomal fractions of the cells. In the cytosol, the best characterized retinol-active dehydrogenases are the dimeric 40 kDa-subunit zinc metalloenzymes that belong to the superfamily of medium-chain alcohol dehydrogenases. Microsomes contain retinol-active dehydrogenases with 35 kDa-subunit molecular weight that belong to the short-chain dehydrogenase/reductase superfamily. Both types of enzymes recognize other physiological substrates besides retinol and exist in multiple isozymic forms. Isozymes exhibit different tissue distribution and have various catalytic efficiencies for oxidation of retinol. The relative physiological importance of the multiple forms of retinol dehydrogenases is currently being debated. This review summarizes some of our data on the identification and characterization of the candidate cytosolic and microsomal retinol dehydrogenases.