Adaptations in fat and carbohydrates metabolism after a prolonged endurance training program were examined using stable isotope tracers of glucose ([6,6-2H2]glucose), glycerol ([2H5]glycerol), and palmitate ([2H2]palmitate). Active, but untrained, males exercised on a cycle for 2 h/day [60% pretraining peak O2 consumption (VO2peak) = 44.3 +/- 2.4 ml.kg-1.min-1] for a total of 31 days. Three cycle tests (90 min at 60% pretraining VO2peak) were administered before training (PRE) and after 5 (5D) and 31 (31D) days of training. Exercise increased the rate of glucose production (Ra) and utilization (Rd) as well as the rate of lipolysis (glycerol Ra) and free fatty acid turnover (FFARa/Rd). At 5D, training induced a 10% (P < 0.05) increase in total fat oxidation because of an increase in intramuscular triglyceride oxidation (+63%, P < 0.05) and a decreased glycogen oxidation (-16%, P < 0.05). At 31D, total fat oxidation during exercise increased a further 58% (P < 0.01). The pattern of fat utilization during exercise at 31D showed a reduced reliance on plasma FFA oxidation (FFA Rd) and a greater dependence on oxidation of intramuscular triglyceride, which increased more than twofold (P < 0.001). In addition, glucose Ra and Rd were reduced at all time points during exercise at 31D compared with PRE and 5D. We conclude that long-term training induces a progressive increase in fat utilization mediated by a greater oxidation of fats from intramuscular sources and a reduction in glucose oxidation. Initial changes are present as early as 5D and occur before increases in muscle maximal mitochondrial enzyme activity.