An improved procedure for the purification of bovine heart mitochondrial pyridine dinucleotide transhydrogenase is described. The enzyme is purified over 100-fold from submitochondrial particles with a 47.4% yield and a specific activity of 62.3 mumol/min/mg. Heart submitochondrial particle membranes were washed with 2 M NaCl to remove peripheral proteins. This was followed by a 1.5% Triton X-100 extraction of the membranes. The extract was then applied to an affinity column of NAD immobilized to agarose and the enzyme eluted with NADH. This step yielded homogeneous enzyme when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation, when reconstituted into phosphatidylcholine liposomes, coupled proton uptake by the vesicles to the reduction of 3-acetylpyridine adenine dinucleotide by NADPH. The polarity characteristics of transhydrogenase were evaluated by the phase separation technique of Bordier (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607); 24% of the enzyme partitioned into the aqueous phase and 76% partitioned into the detergent phase. The amino acid composition was determined and polarity index was calculated to be 40%. These data indicate that the enzyme has hydrophilic as well as hydrophobic characteristics.