Regulation of the activity of the pyruvate dehydrogenase complex in skeletal muscle plays an important role in fuel selection and glucose homeostasis. Activation of the complex promotes disposal of glucose, whereas inactivation conserves substrates for hepatic glucose production. Starvation and diabetes induce a stable increase in pyruvate dehydrogenase kinase activity in skeletal muscle mitochondria that promotes phosphorylation and inactivation of the complex. The present study shows that these metabolic conditions induce a large increase in the expression of PDK4, one of four pyruvate dehydrogenase kinase isoenzymes expressed in mammalian tissues, in the mitochondria of gastrocnemius muscle. Refeeding starved rats and insulin treatment of diabetic rats decreased pyruvate dehydrogenase kinase activity and also reversed the increase in PDK4 protein in gastrocnemius muscle mitochondria. Starvation and diabetes also increased the abundance of PDK4 mRNA in gastrocnemius muscle, and refeeding and insulin treatment again reversed the effects of starvation and diabetes. These findings suggest that an increase in amount of this enzyme contributes to hyperphosphorylation and inactivation of the pyruvate dehydrogenase complex in these metabolic conditions. It was further found that feeding rats WY-14,643, a selective agonist for the peroxisome proliferator-activated receptor-α (PPAR-α), also induced large increases in pyruvate dehydrogenase kinase activity, PDK4 protein, and PDK4 mRNA in gastrocnemius muscle. Since long-chain fatty acids activate PPAR-α endogenously, increased levels of these compounds in starvation and diabetes may signal increased expression of PDK4 in skeletal muscle.