Enzymes catalyzing the two-electron reduction of quinones to hydroquinones are thought to protect the cell against quinone-induced oxidative stress. Using menadione as a substrate, carbonyl reductase, a cytosolic, monomeric oxidoreductase of broad specificity for carbonyl compounds, was found to be the main NADPH-dependent quinone reductase in human liver, whereas DT-diaphorase, the principal two-electron transferring quinone reductase in rat liver, contributed a very minor part to the quinone reductase activity of human liver. Carbonyl reductase from liver was indistinguishable from carbonyl reductase previously isolated from brain (B. Wermuth, J. biol. Chem. 256, 1206 (1981] on the basis of molecular weight, isoelectric point, immunogenicity, substrate specificity and inhibitor sensitivity. The purified enzyme from liver catalyzed the reduction of a great variety of quinones. The best substrates were benzo- and naphthoquinones with short substituents, and the K-region orthoquinones of phenanthrene, benz(a)anthracene, pyrene and benzo(a)pyrene. A long hydrophobic side chain in the 3-position of the benzo- and naphthoquinones and the vicinity of a bay area or aliphatic substituent (pseudo bay area) to the oxo groups of the polycyclic compounds decreased or abolished the ability of the quinone to serve as a substrate. Non-k-region orthoquinones of polycyclic aromatic hydrocarbons were more slowly reduced than the corresponding K-region derivatives. The broad specificity of carbonyl reductase for quinones is in keeping with a role of the enzyme as a general quinone reductase in the catabolism of these compounds.