We report here a mouse cDNA that encodes a 316-amino acid short-chain dehydrogenase that prefers NAD+ as its cofactor and recognizes as substrates androgens and retinols, i.e. has steroid 3alpha- and 17beta-dehydrogenase and cis/trans-retinol catalytic activities. This cis-retinol/androgen dehydrogenase type 2 (CRAD2) shares close amino acid similarity with mouse retinol dehydrogenase isozyme types 1 and 2 and CRAD1 (86, 84, and 87%, respectively). CRAD2 exhibits cooperative kinetics with 3alpha-adiol (3alpha-hydroxysteroid dehydrogenase activity) and testosterone (17beta-hydroxysteroid dehydrogenase activity), but Michaelis-Menten kinetics with androsterone (3alpha-hydroxysteroid dehydrogenase activity), 11-cis-retinol, all-trans-retinol, and 9-cis-retinol, with V/K0.5 values of 1.6, 0.2, 0.1, 0.04, 0.005, and not saturated, respectively. Carbenoxolone (IC50 = 2 microM) and 4-methylpyrazole (IC50 = 5 mM) inhibited CRAD2, but neither ethanol nor phosphatidylcholine had marked effects on its activity. Liver expressed CRAD2 mRNA intensely, with expression in lung, eye, kidney, and brain (2.9, 2, 1.6, and 0.6% of liver mRNA, respectively). CRAD2 represents the fifth isozyme in a group of short-chain dehydrogenase/reductase isozymes (retinol dehydrogenases 1-3 and CRAD1), closely related in primary amino acid sequence (approximately 85%), that are expressed in different quantities in various tissues, have different substrate specificities, and may serve different physiological functions. CRAD2 may alter the amounts of active and inactive androgens and/or convert retinols into retinals. These data expand insight into the multifunctional nature of short-chain dehydrogenases/reductases and into the enzymology of steroid and retinoid metabolism.