Fat and carbohydrate are important fuels for aerobic exercise and there can be reciprocal shifts in the proportions of carbohydrate and fat that are oxidized. The interaction between carbohydrate and fatty acid oxidation is dependent on the intracellular and extracellular metabolic environments. The availability of substrate, both from inside and outside of the muscle, and exercise intensity and duration will affect these environments. The ability of increasing fat provision to downregulate carbohydrate metabolism in the heart, diaphragm and peripheral skeletal muscle has been well studied. However, the regulation of fat metabolism in human skeletal muscle during exercise in the face of increasing carbohydrate availability and exercise intensity has not been well studied until recently. Research in the past 10 years has demonstrated that the regulation of fat metabolism is complex and involves many sites of control, including the transport of fat into the muscle cell, the binding and transport of fat in the cytoplasm, the regulation of intramuscular triacylglycerol synthesis and breakdown, and the transport of fat into the mitochondria. The discovery of proteins that assist in transporting fat across the plasma and mitochondrial membranes, the ability of these proteins to translocate to the membranes during exercise, and the new roles of adipose triglyceride lipase and hormone-sensitive lipase in regulating skeletal muscle lipolysis are examples of recent discoveries. This information has led to the proposal of mechanisms to explain the downregulation of fat metabolism that occurs in the face of increasing carbohydrate availability and when moving from moderate to intense aerobic exercise.