Intramyocellular lipid (IMCL) content has been reported to decrease after prolonged submaximal exercise in active muscle and, therefore, seems to form an important local substrate source. Because exercise leads to a substantial increase in plasma free fatty acid (FFA) availability with a concomitant increase in FFA uptake by muscle tissue, we aimed to investigate potential differences in the net changes in IMCL content between contracting and noncontracting skeletal muscle after prolonged endurance exercise. IMCL content was quantified by magnetic resonance spectroscopy in eight trained cyclists before and after a 3-h cycling protocol (55% maximal energy output) in the exercising vastus lateralis and the nonexercising biceps brachii muscle. Blood samples were taken before and after exercise to determine plasma FFA, glycerol, and triglyceride concentrations, and substrate oxidation was measured with indirect calorimetry. Prolonged endurance exercise resulted in a 20.4 ± 2.8% (P <0.001) decrease in IMCL content in the vastus lateralis muscle. In contrast, we observed a substantial (37.9 ± 9.7%; P <0.01) increase in IMCL content in the less active biceps brachii muscle. Plasma FFA and glycerol concentrations were substantially increased after exercise (from 85 ± 6 to 1,450 ± 55 and 57 ± 11 to 474 ± 54 µM, respectively; P <0.001), whereas plasma triglyceride concentrations were decreased (from 1,498 ± 39 to 703 ± 7 µM; P <0.001). IMCL is an important substrate source during prolonged moderate-intensity exercise and is substantially decreased in the active vastus lateralis muscle. However, prolonged endurance exercise with its concomitant increase in plasma FFA concentration results in a net increase in IMCL content in less active muscle.