Fat and carbohydrate are the principal substrates that fuel aerobic ATP synthesis in skeletal muscle. Most endogenous fat is stored as triacylglycerol in subcutaneous and deep visceral adipose tissue. Smaller quantities of triacylglycerol are deposited as lipid droplets inside skeletal muscle fibers. The potential role of intramyocellular triacylglycerol (IMTG) as a substrate source during exercise in humans has recently regained much of its interest because of the proposed functional relationship between IMTG accumulation and the development of skeletal muscle insulin resistance. Exercise likely represents an effective means to prevent excess IMTG accretion by stimulating its rate of oxidation. However, there is much controversy on the actual contribution of the IMTG pool as a substrate source during exercise. The apparent discrepancy in the literature likely stems from methodological difficulties that have been associated with the methods used to estimate IMTG oxidation during exercise. However, recent studies using stable isotope methodology, 1H-magnetic resonance spectroscopy, and electron and/or immunofluorescence microscopy all support the contention that the IMTG pool can function as an important substrate source during exercise. Although more research is warranted, IMTG mobilization and/or oxidation during exercise seem to be largely determined by exercise intensity, exercise duration, macronutrient composition of the diet, training status, gender, and/or age. In addition, indirect evidence suggests that the capacity to mobilize and/or oxidize IMTG is substantially impaired in an obese and/or Type 2 diabetic state. As we now become aware that skeletal muscle has an enormous capacity to oxidize IMTG stores during exercise, more research is warranted to develop combined exercise, nutritional, and/or pharmacological interventions to effectively stimulate IMTG oxidation in sedentary, obese, and/or Type 2 diabetes patients.