Cardiac fatty acid oxidation (FAO) enzyme gene expression is known to be downregulated during hypoxia in concordance with reduced FAO rates. To evaluate this metabolic switch, the transcriptional control of a cardiac FAO enzyme-encoding gene (medium-chain acyl-CoA dehydrogenase, MCAD) was characterized in response to hypobaric hypoxia. Transgenic mice harboring 560-bp of the human MCAD gene promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene were exposed to moderate (14% O2) or severe (8% O2) hypoxia for 2 or 7 days. MCAD-CAT activity and gene expression were significantly downregulated following 7 days of moderate hypoxia versus normoxic controls (p < 0.05). In parallel two known transcriptional regulators of MCAD expression, PPARalpha and Sp3, were concordantly downregulated at 7 days hypoxia. In contrast, severe hypoxia increased MCAD-CAT activity by 31 +/- 1.4% after 2 days hypoxia, returning to base +/- 4% after 2 days (p < 0.001) and returned to control levels after 7 days of hypoxia. These data demonstrate that MCAD gene expression is downregulated after 7 days of moderate hypoxia and inversely regulated with severe hypoxia. The known MCAD transcriptional regulators PPARalpha and Sp3 mirror MCAD expression. These data indicate that the transcriptional regulatory circuits involved in the control of MCAD gene expression under hypoxic conditions are modulated by upstream factors that are sensitive to the levels of oxygen.