Cytoplasmic malate dehydrogenase has been purified 20 -- fold from the supernatant fraction of chicken liver homogenates in 0.25 M sucrose (differential centrifugation at 100,000 x g) through calcium phosphate gel absorption, ammonium sulphate fractionation (45-80% saturation) and Sephadex G-100 chromatography. Two molecular forms, A and B, of the enzyme are unmodified during the purification process, observed by electrophoresis. Two additional bands that appear in the electrophoretic patterns are not attributable to molecular forms of the enzyme, but to the lack of non-dehydrogenase activity of the preparation used. Both forms differ in their thermal stability (form A is more thermolabile) and net charge; and they both have the same molecular weight (67,000 +/- 5,000 daltons), determined by Sephadex G-200 chromatography. The enzyme in the purified preparations exhibits two KM values in the double reciprocal plot v vs [L-malate], due to the presence of two forms with different affinity for the hydroxyacid.