NADPH-linked alpha,beta-ketoalkene double bond reductase was purified from rat liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose. AF-Blue Toyopearl and hydroxyapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 39,500 by the electrophoresis and by HPLC gel filtration on a TSK gel G3000 SWXL column. The double bond of 2-alkenals was also reduced by the enzyme, but to a lesser extent. The enzyme activity was inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuribenzoic acid, N-ethylmaleimide, iodoacetamide, dicumarol, quercitrin, and disulfirum. However, the enzyme was insensitive to oxygen.