Fatty acids are the most abundant source of endogenous energy substrate. They can be mobilized from peripheral adipose tissue and transported via the blood to active muscle. During higher intensity exercise, triglyceride within the muscle can also be hydrolyzed to release fatty acids for subsequent direct oxidation. Control of fatty acid oxidation in exercise can potentially occur via changes in availability, or via changes in the ability of the muscle to oxidize fatty acids. We have performed a series of experiments to distinguish the relative importance of these potential sites of control. The process of lipolysis normally provides free fatty acids (FFA) at a rate in excess of that required to supply resting energy requirements. At the start of low intensity exercise, lipolysis increases further, thereby providing sufficient FFA to provide energy substrates in excess of requirements. However, lipolysis does not increase further as exercise intensity increases, and fatty acid oxidation becomes approximately equal to the total amount of fatty acids available at 65% of VO2 max. When plasma FFA concentration is increased by lipid infusion during exercise at 85% VO2 max, fat oxidation is significantly increased. Taken together, these observations indicate that fatty acid availability can be a determinant of the rate of their oxidation during exercise. However, even when lipid is infused well in excess of requirements during high-intensity exercise, less than half the energy is derived from fat. This is because the muscle itself is a major site of control of the rate of fat oxidation during exercise. We have demonstrated that the mechanism of control of fatty acid oxidation in the muscle is the rate of entry into the mitochondria. We hypothesize that the rate of glycolysis is the predominant regulator of the rate of carbohydrate metabolism in muscle, and that a rapid rate of carbohydrate oxidation caused by mobilization of muscle glycogen during high intensity exercise inhibits fatty acid oxidation by limiting transport into the mitochondria. During low intensity exercise, glycogen breakdown and thus glycolysis is not markedly stimulated, so the increased availability of fatty acids allows their oxidation to serve as the predominant energy source. At higher intensity exercise, stimulation of glycogen breakdown and glycolysis cause increased pyruvate entry into the TCA cycle for oxidation, and as a consequence the inhibition of fatty acid oxidation by limiting their transport into the mitochondria.