Pig heart DPN-dependent isocitrate dehydrogenase is heterogeneous on isoelectric focusing in 6 M urea. Under these conditions, three types of subunits (termed alpha, beta, and gamma), which have isoelectric points of about 5.7, 6.6, and 7.2, respectively, can be separated. On the basis of densitometric scans of analytical isoelectric focused gels stained with Coomassie blue, it is estiated that the subunits are present in the whole enzyme in the approximate ratio of 2 alpha:1 beta: 1 gamma. The three isolated subunits have distinct amino acid compositions and the amino acid composition of the total enzyme, when expressed as residues per average polypeptide chain of 40,000 daltons, is consistent with contributions of alpha, beta, and gamma subunits in the ratio of 2:1:1. Each isolated subunit yields a readly distinguishable tryptic peptide map which is much simpler than that of the total enzyme, and is consistent with the number of peptides expected from the lysyl plus arginyl residues for the subunit. The alpha and beta chains both have alanine as the NH2-terminal amino acid, whereas phenylalanine is the NH2-terminal residue of the gamma subunit. Since the alpha subunit exhibits a molecular weight of 39,000 and the beta and gamma subunits have indistinguishable molecular weights of 41,000, the two-band pattern observed on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is understandable. These results suggest that a complete DPN-specific isocitrate dehydrogenase would have a minimum molecular weight of 160,000.