We tested the hypothesis that hypoxia decreases PPARα-regulated gene expression in heart muscle in vivo. In two rat models of systemic hypoxia (cobalt chloride treatment and iso-volemic hemodilution), transcript levels of PPARα and PPARα-regulated genes (pyruvate dehydrogenase kinase 4 (PDK4), muscle carnitine palmitoyltransferase-I (mCPT-I), and malonyl-CoA decarboxylase (MCD)) were measured using real-time quantitative RT-PCR. Data were normalized to the housekeeping gene β-actin. Atrial natriuretic factor (ANF) and pyruvate dehydrogenase kinase 2 (PDK2), which are not regulated by PPARα, served as controls. CoCl₂ treatment decreased PPARα, PDK4, mCPT-I, and MCD mRNA levels. Iso-volemic anemia also caused a significant decrease in PPARα, PDK4, and MCD mRNA levels. Transcript levels of mCPT-I showed a slight, but not significant decrease (P = 0.08). Gene expression of β-actin, ANF, and PDK2 did not change with either CoCl₂ treatment nor with anemia. Myocardial PPARα-regulated gene expression is decreased in two models of hypoxia in vivo. These results suggest a transcriptional mechanism for decreased fatty oxidation and increased reliance of the heart for glucose during hypoxia.