Mitochondrial glycerol-3-P dehydrogenase (EC 1.1.99.5) has been purified in 20% yield from both rabbit skeletal muscle and brain using a four step procedure involving osmotic shock, solubilization with Triton X-100, hydrophobic chromatography, gel filtration, and preparative column isoelectrofocusing. The active muscle and brain enzymes were found to be 95% and 80% homogeneous, respectively. Final purification was performed on the denatured subunit. The active enzyme from each of the tissues focused at pH 5.25 +/- 0.12 and each produced similar biphasic thermal inactivation plots at 50 degrees C. Mixtures of the purified brain and muscle enzymes co-migrated in discontinuous electrophoresis gels and each enzyme exhibited a single polypeptide component on sodium dodecyl sulfate (SDS) gels either when run separately or in mixtures. The subunit molecular weight was shown to be 76,000 +/- 3,000 by SDS-gel electrophoresis and gel filtration in 6 M guanidine HCl. One mole of noncovalently bound FAD and 1 mole of iron were measured per Mr = 100,000. The amino acid composition was determined based on the assumption of 70 aspartate residues per subunit to give a Mr = 76,000. The absorption spectrum has a maximum at 416 nm and a shoulder at 450 to 460 nm which is bleached on treatment with sodium dithionite. The maximum at 416 nm is removed by treatment with mersalyl.