Intramyocellular triacylglycerol (IMTG) has been shown to be an important substrate source during exercise. The present study assessed IMTG utilization via 3 methodologies. In addition, we identified differences in the use of IMTG deposited in the immediate subsarcolemmal area (SS) versus IMTG stored in the central region of the fiber. Stable isotopes were applied in combination with muscle tissue sampling before and immediately after 3 h of cycling (62±2% VO_2max) in 8 well-trained males. Continuous infusions with [U-¹³C] palmitate and [6,6-²H₂] glucose were applied to quantify plasma free fatty acid (FFA) and glucose oxidation rates and to estimate whole-body IMTG and glycogen use. Immunohistochemical analyses of oil red O (ORO) stained muscle cross-sections and biochemical triacylglycerol (TG) extraction were performed to assess muscle lipid content. During exercise, plasma FFA, muscle derived TG, plasma glucose and muscle glycogen oxidation contributed 24±2, 22±3, 11±1 and 43±3% to total energy expenditure, respectively. In accordance, a significant net decline in muscle lipid content was observed following exercise as assessed by ORO staining (67±8%) and biochemical TG extraction (49±8%), and a positive correlation was observed between methods (R=0.56; p<0.05). Lipid depots located in the SS area were utilized to a greater extent versus centrally located depots. This is the first study to show significant use of IMTG during exercise in healthy males via the concurrent implementation of 3 major methodologies. This study also shows differences in resting subcellular IMTG deposit distribution and in the subsequent net use of these deposits during exercise.