Pyruvate dehydrogenase kinase (PDK) catalyzes phosphorylation and inactivation of the pyruvate dehydrogenase complex (PDC). Two isoforms of this mitochondrial kinase (PDK2 and PDK4) are induced in a tissue-specific manner in response to starvation and diabetes. Inactivation of PDC by increased PDK activity promotes gluconeogenesis by conserving three-carbon substrates. This helps maintain glucose levels during starvation, but is detrimental in diabetes. Factors that regulate PDK2 and PDK4 expression were examined in Morris hepatoma 7800 C1 cells. The peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist WY-14,643 and the glucocorticoid dexamethasone increased PDK4 mRNA levels. Neither compound affected the half-life of the PDK4 message, suggesting that both increase gene transcription. Fatty acids caused an increase in the PDK4 message comparable to that induced by WY-14,643. Insulin prevented and reversed the stimulatory effects of dexamethasone on PDK4 gene expression, but was less effective against the stimulatory effects of WY-14,643 and fatty acids. Insulin also decreased the abundance of the PDK2 message. The findings suggest that decreased levels of insulin and increased levels of fatty acids and glucocorticoids promote PDK4 gene expression in starvation and diabetes. The decreased level of insulin is likely responsible for the increase in PDK2 mRNA level in starvation and diabetes.