Both pyruvate dehydrogenase (PDH) kinase and PDH phosphatase require a divalent cation for activity. Either Mg 2+ or Mn 2+, but not Ca 2+, can satisfy this requirement. The apparent K m of the kinase for MgATP 2− is about 0.02 m m, whereas the apparent K m of the phosphatase for Mg 2+ is about 2 m m. The apparent K m of the phosphatase for Mn 2+ is about 0.5 m m. The bovine kidney and heart PDH kinases exhibit pH optima about 7.0–7.2 in the presence of either Mg 2+ or Mn 2+. The pH optima of the corresponding PDH phosphatases are about 6.7–7.1 in the presence of Mg 2+ and 7.5–7.6 in the presence of Mn 2+. ADP is a competitive inhibitor of ATP. The apparent K i for ADP is about 0.1 m m. No effect of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) on either the kinase or the phosphatase was observed in these studies. Inhibition of PDH phosphatase by fluoride ion, inorganic orthophosphate, EDTA and EGTA is described. Pyruvate protects the pyruvate dehydrogenase complex against inactivation by ATP, and this effect appears to be more pronounced with the bovine heart complex than with the bovine kidney complex. It appears that pyruvate exerts its inhibitory effect primarily on the PDH kinase. We have observed that the kidney kinase catalyzes a phosphorylation of casein. This reaction is insensitive to cyclic AMP, and it is inhibited by pyruvate and α-ketobutyrate, but not by α-ketoglutarate. Dihydrolipoyl transacetylase markedly stimulates the rate of phosphorylation of PDH by PDH kinase. We have found that the transacetylase lowers the apparent K m of the kinase for PDH from about 20 μ m to about 0.6 μ m.