We have recently shown that increased carbohydrate flux decreases fat oxidation during exercise by inhibition of fatty acid entry into the mitochondria. Because endurance training reduces the rate of carbohydrate flux during exercise, we hypothesized that training increases fat oxidation by relieving this inhibition. To test this hypothesis, five sedentary and five endurance-trained men exercised on a cycle ergometer at an oxygen consumption (VO2) of approximately 2.0 l/min, representing 80 and 40% peak VO2, respectively. [1-13C]oleate and [1-14C]octanoate, long- and medium-chain fatty acids, respectively, were infused for the duration of the studies. Carbohydrate oxidation was significantly higher in the sedentary group (196 +/- 9 vs. 102 +/- 17 mumol.kg-1.min-1, P < 0.05). Oleate oxidation was higher in the trained group (3.8 +/- 0.6 vs. 1.9 +/- 0.3 mumol.kg-1.min-1, P < 0.05), whereas octanoate oxidation was not different between the two groups. The percentage of oleate that was taken up by tissues and oxidized was higher in the trained group (76 +/- 7 vs. 58 +/- 3%, P < 0.05). However, the percentage of octanoate taken up and oxidized was not different (82 +/- 3 vs. 85 +/- 4%, not significant). Because octanoate, unlike oleate, can freely diffuse across the mitochondrial membrane, the present results suggest that the difference in fatty acid oxidation between trained and untrained individuals may be due to enhanced fatty acid entry into the mitochondria.