Beta-adrenergic receptor (beta-AR) modulation of resting and postprandial energy expenditure (EE) is augmented in regularly exercising compared with sedentary adults, but the underlying physiological mechanisms are unknown. Differences in thermogenic responsiveness to beta-AR stimulation, perhaps secondary to reactive oxygen species (ROS) bioactivity, may be involved. To determine habitual exercise-related differences in beta-AR thermogenic responsiveness and the possible influence of ROS, we measured the percentage increase in EE (DeltaEE%; indirect calorimetry, ventilated hood method) above resting EE in response to non-specific beta-AR stimulation (intravenous isoproterenol (isoprenaline): 6, 12 and 24 ng (kg fat-free mass)-1 min-1) in 25 sedentary (11 males; 51+/-4 years; body mass index 25.0+/-0.8 kg m-2, maximal oxygen uptake 29+/-1 ml kg-1 min-1 (mean+/-s.e.m.)) and 14 habitually aerobic exercising (9 males, 46+/-6 years, 23.1+/-0.7 kg m-2, 44+/-3 ml kg-1 min-1) healthy adults under normal (control) conditions and during acute intravenous administration of a potent antioxidant, ascorbic acid (vitamin C; 0.04 g (kg fat-free mass)-1). DeltaEE% was greater (P=0.02) in the habitually exercising (8.6+/-1.2, 12.9+/-1.2, 20.0+/-1.4) versus sedentary (6.3+/-0.7, 10.4+/-0.8, 16.0+/-1.0) adults. Ascorbic acid increased (P=0.01) DeltaEE% only in the sedentary adults (to 9.5+/-0.9, 12.4+/-0.7, 18.5+/-0.8), abolishing baseline group differences. DeltaEE% was not related to the amount of body fat, sex, or any other baseline characteristic. Thermogenic responsiveness to beta-AR stimulation is augmented in habitually exercising adults. The mechanism is ascorbic acid dependent, suggesting that it may be linked to decreased ROS bioactivity. Our findings advance a novel mechanism by which habitual physical activity may modulate EE in humans, with potential implications for energy balance and body weight control.