Chicken heart mitochondrial malate dehydrogenase has been purified by an improved isolation method to give 56% of the initial mitochondrial enzyme. The purified enzyme has a specific activity of 340 U/mg. This homogeneous enzyme has been shown to be pure by several criteria. The enzyme has been shown to be a dimer with a molecular weight of 67,000 gm/mol. Upon exposure of MDH to pH 4.8, the enzyme dissociates into 33,000 gm/mol monomers. At pH 5.0, an apparent equilibrium. exists between the monomeric and dimeric states. If pyrophosphate or phosphate were present, stabilization of the enzyme occurred, causing an increase in the enzymatic activity and a decrease in the K_m,values for both substrate and coenzyme, probably by an induced conformational change. Iodoacetamide was found to modify two histidine residues, one group in each active site, per enzyme dimer at pH 7.0. Ellman's reagent reacted with enzyme at pH 5.0, possibly modifying most of the sulfhydryl groups. N-Ethyl maleimide modification of the enzyme's sulfhydryl groups at pH 4.8 was found to be slightly more specific in its modification than Ellman's reagent. The number of sulfhydryl groups modified by N-ethyl maleimide increased with either lowering of the pH or an increased reagent concentration. Six -SH groups were modified at pH 5.0, while eleven sulfhydryl groups reacted at pH 4.8. The presence of NADH was found to prevent modification entirely when either N-ethyl maleimide or iodoacetamide was present. A model of subunit interactions in the native and modified chicken isozyme is presented, and is compared with the model of the porcine and bovine isozymes. / M.S.