The activity of the pyruvate dehydrogenase kinase, which phosphorylates and thereby inactivates the pyruvate dehydrogenase complex, was stimulated by malonyl-CoA. Treatment with [2-14C]malonyl-CoA resulted in acylation of sites in the complex. Both acylation and activation of kinase activity increased in a time-dependent manner with a parallel increase in those activities when the malonyl-CoA:CoA ratio was varied. Protein-bound acyl groups were labilized by performic acid treatment indicating their attachment to protein at thiol residues; however, the product released was volatile, which is not characteristic of malonic acid. While malonyl-CoA was initially free of acetyl-CoA, stimulation of kinase activity and acylation of sites in the complex by malonyl-CoA were shown to be contingent upon enzyme-catalyzed decarboxylation. Decarboxylation appeared to be catalyzed by a trace contaminant present in highly purified preparations of both the pyruvate and 2-oxoglutarate dehydrogenase complexes. Under conditions in which both free CoA was removed (by conversion to succinyl-CoA) and then, after various periods, free acetyl-CoA was removed (by enzymic conversion to acetyl phosphate), both acetylation of sites in the complex and activation of kinase activity increased in a time-dependent manner. Concomitantly there was a decrease in the concentration dependence for activation of the kinase by malonyl-CoA. Our results strongly support the conclusion that activation of kinase activity is associated with acylation of sites in the complex, and that, in the case of malonyl-CoA, those processes depend on enzyme-catalyzed decarboxylation.