1. The influence of training-induced adaptations in skeletal muscle tissue on the choice between carbohydrates (CHO) and lipids as well as the extra- vs. intracellular substrate utilization was investigated in seven healthy male subjects performing one-legged knee-extension exercise. In each subject one of the knee extensors was endurance trained for eight weeks, whereafter the trained (T) and non-trained (NT) thighs were investigated a week apart. 2. The activity of beta-hydroxy-acyl-coenzyme A dehydrogenase (HAD) and capillary density in the knee extensors were significantly larger in T than in NT. 3. During dynamic knee-extension exercise, performed at the same absolute intensity for 2 h, femoral venous blood flow was lower in T than in NT (P < 0.05), but oxygen uptake was similar. 4. Respiratory quotient (RQ) values over the exercising thigh, averaging 0.81 (T) vs. 0.91 (NT; P < 0.05) indicated that a shift towards a larger fat combustion occurred with endurance training. 5. Both free fatty acids (FFA) and serum triacylglycerol contributed to the utilization of fat in NT and T muscles with no significant contribution from muscle fibre triacylglycerol. 6. At high plasma FFA concentrations net uptake of FFA plateaued in NT but not in T muscles. 7. The findings suggest that FFA uptake in exercising muscle is a saturable process and that the transport capacity is enhanced by training. The lower CHO utilization in the T leg was mainly a function of the glycogenolysis of the muscle being reduced. Hormones such as insulin, noradrenaline and adrenaline are unlikely to play a role in this shift as differences in plasma levels during T and NT leg exercise were small and insignificant, implying that local structural and functional adaptations of the training muscle are crucial for the observed shifts in the metabolic response to exercise.