OBJECTIVE Fat metabolism is increasingly implicated in the pathogenesis of type 2 diabetes. Endurance training has been shown to prevent hepatic steatosis and to alter skeletal muscle fat metabolism, and regional free fatty acid (FFA) uptake adaptations were suggested as a mechanism. Thus, we tested whether endurance training modifies the uptake of plasma FFAs occurring in the liver and in skeletal muscle during anabolic, i.e., hyperinsulinemic, conditions. RESEARCH DESIGN AND METHODS Trained and untrained healthy male subjects underwent positron emission tomography scanning of the liver and thigh regions, with the FFA analog 14(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid, during euglycemic hyperinsulinemia. Tracer influx rate constants in skeletal muscle (MK(i)) and liver (LK(i)) were multiplied by plasma FFA levels to obtain FFA uptake for skeletal muscle (MFU) and liver (LFU), respectively. RESULTS Athletes showed increased Vo(2max) (P < 0.0001), insulin-mediated glucose disposal (M value, 61 +/- 4 vs. 46 +/- 3 micromol. min(-1). kg(-1), P = 0.01), and plasma lactate levels during the clamp and lower percentage of body fat mass (P = 0.002). MK(i) was 25% higher in athletes than in sedentary men (P = 0.03). In all subjects, MK(i) and MFU were positively correlated with the M value (r = 0.56, P = 0.02, and r = 0.51, P = 0.03, respectively) and with plasma lactate levels (r = 0.63, P = 0.006, and r = 0.63, P = 0.005, respectively). LK(i) was significantly reduced by 20% in the athletes (P = 0.04). By multiple regression, LFU was inversely correlated with the two fitness categories (P = 0.008), and it was lower in athletes. Linear fitting of liver data showed time consistency, indicating no release of FFAs as a mechanism for the reduced liver retention in athletes. CONCLUSIONS We conclude that endurance training promotes insulin-mediated glucose and FFA disposal in skeletal muscle, while lowering hepatic FFA uptake. Such changes may result in a divergent pattern of fat accumulation in the two organs.