The hearts obtains 70% of its energy for contraction from the oxidation of fatty acids, derived from adipose, hepatic and dietary sources. Myocardial ischemia is accompanied by increased adipose lipolysis and elevated fatty acid supply to the heart. We investigated the effects of acute and chronic ischemia on cardiac fatty acid uptake and metabolism. Wistar rat hearts were perfused in Langendorff contracting mode during pre-ischemia, low-flow ischemia and reperfusion, using 3H-substrates for measurement of substrate metabolism. During 30 mins of ischemia, there was a 32% decrease in sarcolemmal location of the predominant fatty acid transporters, FAT/CD36, as it translocated into intracellular endosomes to limit cardiac fatty acid uptake. This was accompanied by a 95% decrease in fatty acid oxidation rates, with no change in intramyocardial lipids. Following 30 mins of reperfusion, decreased sarcolemmal FAT/CD36 persisted, but fatty acid oxidation rates returned to pre-ischemic levels in line with contractile function, but resulted in a 35% decrease in myocardial triglyceride content. To investigate the longer term metabolic effects of ischemia, changes in cardiac fatty acid metabolism were investigated in the chronically ischemic rat heart, 6 months after in vivo myocardial infarction. Fatty acid oxidation and myocardial lipid incorporation were decreased by 30% and 25%, respectively, correlating positively with the in vivo cardiac ejection fraction. In contrast to acute ischemia, chronic ischemia decreased total protein levels of FAT/CD36, accompanied by decreased levels of other fatty acid transporters and fatty acid oxidation proteins. In conclusion, during acute ischemia, FAT/CD36 moved away from the sarcolemma, associated with a shift from fatty acid oxidation to glycolysis, whilst intramyocardial lipid accumulation was prevented. In contrast, following chronic ischemia, fatty acid utilisation is downregulated, achieved by downregulating multiple proteins involved in fatty acid metabolism. In light of the increased adipose lipolysis during acute and chronic ischemia, the cardiac metabolic changes are likely a protective response to prevent fatty acid uptake under conditions of limited oxidation.