The influence of glucose ingestion on substrate utilization during prolonged exercise in children and adolescents is currently unknown. In the present study we determined the effect of intermittent exogenous glucose (GLUexo) ingestion on substrate utilization during prolonged exercise, in adolescent boys ages 13 17 years. Healthy untrained volunteers performed four 30-min exercise bouts on a cycle ergometer, separated by 5-min rest periods (approximately equal to 60% maximum O2 consumption), on two occasions spaced 1-4 weeks apart. Two trials were performed, a control trial (CT), in which subjects ingested water intermittently during the exercise, and a glucose trial (GT), in which subjects ingested a 13C-enriched GLUexo drink (approximately egual to glucose kg body mass(-1)), also intermittently during the exercise. Total free fatty acids (FATtotal), glucose (GLUtotal) and carbohydrate (CHOtotal) oxidation was determined from indirect calorimetry, while GLUexo oxidation was calculated from the 13C/12C ratio in expired air after 5-10 min and 25-30 min of exercise in each bout. Heart rate and rating of perceived exertion (RPE) were determined at the same time intervals. The oxidation of CHOtotal was 169.1 (12.9) g x 120 min(-1) and 203.1 (15.9) g x 120 min(-1) (P < 0.01) and that of FATtotal was 31.0 (4.2) g x 120 min(-1) and 17.1 (2.5) g x 120 min(-1) (P < 0.01) in CT and GT, respectively. GLUexo oxidation in GT was 57.8 (4.3) g x 120 min(-1), or 34.2 (2.2)% of that ingested. Endogenous glucose oxidation was 169.1 (12.9) g x 120 min(-1) and 145.3 (11.9) g x 120 min(-1) (P < 0.01) in CT and GT, respectively. Insulin and glucose concentrations were higher in GT than in CT by 226% and 37%, respectively (both P < 0.05). Free fatty acids and glycerol concentrations were lower in GT than in CT, by 27% and 79%, respectively (both P < 0.05). Heart rate was similar between trials, but RPE was lower in GT vs CT at both 115 and 135 min. Thus, under these experimental conditions, GLUexo intake spares endogenous carbohydrate and fat by 16% and 45%, respectively, contributes to approximately 25% of the total energy demand of exercise, and lowers the RPE.