Hypoxia triggers a cascade of cellular energy metabolic responses including a decrease in mitochondrial oxidative flux. To characterize gene regulatory mechanisms by which mitochondrial fatty acid oxidative capacity is diminished in response to hypoxia, cardiac myocytes in culture were exposed to long-chain fatty acids (LCFA) under normoxic or hypoxic conditions. Hypoxia prevented the known LCFA-induced accumulation of mRNA encoding muscle carnitine palmitoyltransferase I (M-CPT I), an enzyme that catalyzes the rate-limiting step in mitochondrial fatty acid oxidation (FAO). Under hypoxic conditions, myocytes exhibited significant accumulation of intracellular neutral lipid consistent with reduced CPT I activity and diminished FAO capacity. Transient transfection experiments demonstrated that the hypoxia-mediated blunting of M-CPT I gene expression occurs at the transcriptional level, is localized to an LCFA/peroxisome proliferator-activated receptor alpha (PPARalpha)/retinoid X receptor (RXR) response element within the M-CPT I gene promoter, and is PPARalpha-dependent. DNA-protein binding studies demonstrated that exposure to hypoxia reduces PPARalpha/RXR binding activity. Immunoblotting studies demonstrated that whereas hypoxia had no effect on nuclear levels of PPARalpha protein, nuclear and cellular RXRalpha levels were reduced. Hypoxia also diminished the 9-cis-retinoic acid-mediated activation of a reporter containing an RXR homodimer response element. These results demonstrate that hypoxia deactivates PPARalpha by reducing the availability of its obligate partner RXR.