An enzyme catalyzing the metabolic reduction of acetohexamide, an oral antidiabetic drug, has been purified from the cytosolic fraction of rabbit kidney to apparent homogeneity by various chromatographic techniques. The purified enzyme consists of a single polypeptide chain with a molecular weight of 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme requires NADPH as a cofactor and has an optimal pH of 6.0. A variety of xenobiotic carbonyl compounds including acetohexamide are effectively reduced by the enzyme. Flavonoids (quercetin and quercitrin) are potent inhibitors for the enzyme, but pyrazole or barbiturates have little effect on the enzyme activity. These findings clearly indicate that the enzyme can be classified as one of the carbonyl reductases. The enzyme also shows both prostaglandin 9-ketoreductase and 3 alpha-hydroxysteroid dehydrogenase activities. Judging from the Kcat/Km values of the enzyme for 4-pyridylketones with a straight-chain alkyl group, a hydrophobic pocket that binds most strongly to a straight-chain alkyl group of five carbon atoms in length appears to be located in the substrate-binding region of the enzyme.