Phosphorylation of the alpha-subunits of the branched chain alpha-keto acid and the pyruvate dehydrogenases was measured in mitochondria isolated from rat hearts perfused in the presence of [32P]phosphate and various substrates. Mitochondrial isolations were accomplished rapidly under conditions which would preclude the interconversion of these two enzyme complexes between their active (dephosphorylated) and inactive (phosphorylated) forms. Inactivation of the branched chain complex and the concomitant incorporation of [32P]phosphate into the alpha-chain of the dehydrogenase subcomponent were observed in hearts perfused with glucose (10 mM) or pyruvate (10 mM). alpha-Ketoisocaproate infusion caused a 15-fold activation and dephosphorylation of the branched chain dehydrogenase, while pyruvate dehydrogenase remained inactive and phosphorylated under these conditions. Both dehydrogenase components were partially activated, and most of the [32P]phosphate was removed from their respective alpha-subunits during substrate-free perfusion. Both enzyme complexes were activated and dephosphorylated completely during infusion of dichloroacetate (1 mM) and glucose (10 mM).