The three dissimilar subunits of pig heart DPN-dependent isocitrate dehydrogenase, which normally occur in the ratio 2 alpha:1 beta:1 gamma, have been separated by chromatofocusing. The subunits were first dissociated from native enzyme (specific activity, 15-20 units/mg) with minimal unfolding in 2 M urea and chromatofocused in the range pH 8-5 in the presence of 1 M urea. Isolated alpha, beta, and gamma subunits had specific activities of 0.4, 0.04, and less than 0.01 unit/mg, respectively, suggesting that at least 3 of the 4 subunits of the alpha 2 beta gamma tetramer contain elements of the catalytic site. Addition of pairs of subunits to assay mixtures resulted in markedly enhanced activity for alpha plus gamma (specific activity, 7.8 units/mg) and alpha plus beta (2.9 units/mg), but not for beta plus gamma. The subunit concentration dependence of enhanced activity indicated formation of stoichiometric alpha beta and alpha gamma complexes. Sedimentation equilibrium experiments revealed that alpha, as isolated, exists primarily as a dimer, while beta and gamma are monomeric. The combinations of alpha and beta and alpha and gamma are dimers. Under the conditions used, no larger complexes were formed when all 3 subunits were added in the ratio (2 alpha:1 beta:1 gamma) found in native enzyme and these mixtures failed to show increased activity above that found with alpha beta and alpha gamma. Native enzyme exhibits a reduced Km for the substrate, isocitrate, in the presence of ADP, but this allosteric effect was not observed for the isolated subunits or for combinations of alpha with beta or gamma. However, ADP binding to isolated alpha and beta subunits as well as to alpha beta and alpha gamma dimers was only slightly reduced from that of native enzyme. These results indicate that although all subunits may have the elements of the catalytic and allosteric sites, optimum activity and regulatory properties may require the association of dissimilar subunits.