L-Threonine dehydrogenase was purified 10,000-fold to a specific activity approximately 300 mumol.min-1.mg-1 protein from porcine liver mitochondria. Purification to apparent homogeneity was achieved by sequential chromatography on DEAE Sepharose FF, Affi-Gel Blue, Sephacryl S-200, Matrex Gel Red A, and Matrex Gel Green A. The subunit molecular mass was estimated as 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while an apparent native molecular mass of 73 kDa was shown by gel filtration chromatography, suggesting a dimeric structure. The purified enzyme was subjected to proteolytic degradation and the resulting peptides were isolated exclusively by reverse-phase high-performance liquid chromatography. Approximately 70% of the total sequence was obtained and the N-terminal amino acid sequence of the intact polypeptide chain was thus tentatively extended to residue 40.