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 all-trans-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 3α-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 μM, respectively. All-trans- retinol bound to cellular retinol-binding protein (type I) exhibits a similar K(i) value of 3.6 μM. 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 3α-androstane-diol.