The mitochondrial kinases responsible for the phosphorylation and inactivation of rat heart pyruvate dehydrogenase complex and the rat liver and heart branched-chain α-ketoacid hydrogenase complexes have been purified to homogeneity. The branched-chain α-ketoacid dehydrogenase kinase is composed of one subunit with a molecular weight of 44 kDa; pyruvate dehydrogenase kinase has two subunits with molecular weights of 48 (α) and 45 kDa (β). Proteolysis maps of branched-chain α-ketoacid dehydrogenase kinase and the two subunits of pyruvate dehydrogenase kinase are different, suggesting that all subunits are different entities. The α subunit of the rat heart pyruvate dehydrogenase kinase was selectively cleaved by chymotrypsin with concomitant loss of kinase activity, as previously shown for the bovine kidney enzyme, suggesting that the catalytic activity of pyruvate dehydrogenase kinase resides in this subunit. Polyclonal antibodies against branched-chain α-ketoacid dehydrogenase kinase, purified by an epitope selection method, bound only to the 44 kDa polypeptide of the branched-chain α-ketoacid dehydrogenase complex, substantiating that the 44 kDa protein corresponds to the kinase for this complex. Both kinases exhibited strong substrate specificity toward their respective complexes and would not inactivate heterologous complexes. The kinases possessed slightly different substrate specificities toward histones. Phosphorylation and inactivation of the branched-chain α-ketoacid dehydrogenase complex by its purified kinase was inhibited by α-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the complex. cDNAs encoding the branched-chain α-ketoacid dehydrogenase kinase have been isolated from rat heart and rat liver λgt11 libraries. This represents the first successful cloning of a mitochondrial protein kinase. Preliminary data suggest that two different isoforms of the kinase may exist in different ratios in various tissues. No evidence was found for induction of the branched-chain α-ketoacid dehydrogenase complex nor its kinase by clofibric acid. Rather, clofibric acid is a potent inhibitor of the activity of the branched-chain α-ketoacid dehydrogenase kinase and this may be the molecular mechanism responsible for the myotonic effects of clofibric acid in man. © 1992.