We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/ reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat alltrans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (<73% identity). The mRNA for RoDH-4 is abundant in adult liver, where it is translated into RoDH-4 protein, which is associated with microsomal membranes, as evidenced by Western blot analysis. Significant amounts of RoDH-4 message are detected in fetal liver and lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3␣-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD ؉ and NADH are the preferred cofactors, with apparent K m values 250 -1500 times lower than those for NADP ؉ and NADPH. All-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent K i values of 5.8 and 3.5 M, respectively. All-transretinol bound to cellular retinol-binding protein (type I) exhibits a similar K i value of 3.6 M. Unliganded cellular retinol-binding protein has no effect on RoDH activity. Citral and acyclic isoprenoids also act as inhibitors of RoDH-4 activity. Ethanol is not inhibitory. Thus, we have identified and characterized a sterol/retinol-oxidizing short chain dehydrogenase/reductase that prefers NAD ؉ and recognizes all-trans-retinol as substrate. RoDH-4 can potentially contribute to the biosynthesis of two powerful modulators of gene expression: retinoic acid from retinol and dihydrotestosterone from 3␣-androstane-diol.Short chain alcohol dehydrogenases/reductases are either cytosolic or membrane-bound enzymes with a subunit molecular mass of 25-35 kDa that utilize a vast variety of substrates, including steroids and prostaglandins (1). Recently, this family of enzymes has expanded to include the retinol-oxidizing dehydrogenases (2-6). Retinol dehydrogenases are involved in the biosynthesis of all-trans-retinoic acid, the activating ligand for a family of nuclear receptors (7). All-trans-retinoic acid is produced from all-trans-retinol in two oxidative steps: all-transretinol is oxidized to all-trans-retinal and then further to alltrans-retinoic acid. Retinol dehydrogenases catalyze the ratelimiting step: the oxidation of retinol to retinaldehyde (8). Although the effects of retinoic acid on gene transcription and regulation have been intensively studied during the last decade, the exact enzymes that synthesize this morphogen and the mechanisms that regulate its production in tissues are not fully understood. Enzymatic activity capable of oxidizing retinol to retinaldehyde is readily detected in the cytosolic and microsomal fractions of total cell homogenates (9). The cytosol...