Whole body lipid kinetics were evaluated during basal resting conditions, 4 h of treadmill exercise eliciting an oxygen uptake of 20 ml.kg-1.min-1, and 1 h of recovery in five untrained and five endurance-trained men. Glycerol and free fatty acid (FFA) rate of appearance (Ra) values in plasma were determined by infusing [2H5]glycerol and [1-13C]palmitate, respectively, and lipid oxidation was determined by indirect calorimetry. The lipolytic response to 4 h of exercise, expressed as the average glycerol and FFA Ra values, was similar in both trained (9.85 +/- 1.02 and 24.64 +/- 3.76 mumol.kg-1.min-1, respectively) and untrained subjects (11.29 +/- 0.99 and 24.13 +/- 0.39 mumol.kg-1.min-1, respectively). However, mean triglyceride oxidation was greater during exercise in the trained than in the untrained group (7.51 +/- 0.26 and 5.67 +/- 0.51 mumol.kg-1.min-1, respectively; P < 0.001). During recovery, glycerol and FFA Ra values decreased more rapidly in trained than in untrained subjects. We conclude that highly trained male endurance runners use more fat as a fuel during low-intensity exercise than do untrained healthy men despite similar rates of lipolysis and FFA uptake from plasma. Therefore, the increase in fat oxidation must be related to an increased percentage of FFA uptake oxidized, a greater contribution from intramuscular triglyceride stores, or both. Additionally, lipid kinetics return to baseline more rapidly in trained than in untrained subjects after completing an exercise bout of the same absolute intensity.