The transcription of key metabolic regulatory enzymes in the heart is altered in the diabetic state, yet little is known of the underlying mechanisms. The aim of this study was to investigate the role of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in modulating cardiac insulin-sensitive glucose transporter (GLUT-4) protein levels in altered metabolic states and to determine the functional consequences by assessing cardiac ischemic tolerance. Wild-type and PPAR-alpha-null mouse hearts were isolated and perfused 6 wk after streptozotocin administration or after 14 mo on a high-fat diet or after a 24-h fast. Myocardial d-[2-(3)H]glucose uptake was measured during low-flow ischemia, and differences in GLUT-4 protein levels were quantified using Western blotting. In wild-type mice in all three metabolic states, elevated plasma free fatty acids were associated with lower total cardiac GLUT-4 protein levels and decreased glucose uptake during ischemia, resulting in poor postischemic functional recovery. Although PPAR-alpha-null mice also had elevated plasma free fatty acids, they had neither decreased cardiac GLUT-4 levels nor decreased glucose uptake during ischemia and, consequently, did not have poor recovery during reperfusion. We conclude that elevated plasma free fatty acids are associated with increased injury during ischemia due to decreased cardiac glucose uptake resulting from lower cardiac GLUT-4 protein levels, the levels of GLUT-4 being regulated, probably indirectly, through PPAR-alpha activation.