Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes a pivotal reaction in mitochondrial fatty acid (FA) beta-oxidation. To examine the potential role of FAs and their metabolites in the regulation of MCAD gene expression, we measured MCAD mRNA levels in animals fed inhibitors of mitochondrial long-chain FA import. Administration of carnitine palmitoyltransferase I inhibitors to mice or rats resulted in tissue-limited increases in steady-state MCAD mRNA levels. HepG2 cell cotransfection experiments with MCAD promoter reporter plasmids demonstrated that this was a transcriptional effect mediated by the peroxisome proliferator-activated receptor (PPAR). The activity mapped to a nuclear receptor response element that functioned in a heterologous promoter context and specifically bound immunoreactive PPAR in rat hepatic nuclear extracts, confirming an in vivo interaction. PPAR-mediated transactions of this promoter and element were also induced by exogenously added FA and fibric acid derivatives. Induction of PPAR transactivation by perturbation of this discrete metabolic step is unusual and indicates that intracellular FA metabolites that accumulate during such inhibition can regulate MCAD expression and are likely candidates for PPAR ligand(s). These results dictate an expanded role for the PPAR in the regulation of FA metabolism.