In a randomized double-blind crossover design, we investigated the effect of the beta2-agonist terbutaline on endurance performance and substrate utilization in nine moderately trained males (maximum oxygen uptake (VO2max): 58.9±3.1 mL min(-1) kg(-1)). Subjects performed 60 min of submaximal exercise (65-70% of VO2max) immediately followed by a 300-kcal time trial with inhalation of either terbutaline (TER) or placebo (PLA). Pulmonary gas exchange was measured during the submaximal exercise and muscle biopsies were collected before and after the exercise bouts. Time trial performance was not different between PLA and TER (1054±125 vs. 1072±145 s). During the submaximal exercise, respiratory exchange ratio, glycogen breakdown (PLA: 195±28; TER: 266±32 mmol kg dw(-1)) and muscle lactate accumulation (PLA: 13.2±1.2; TER: 20.3±1.6 mmol kg dw(-1)) were higher (P<0.05) with TER than PLA. There was no difference between PLA and TER in net muscle glycogen utilization and lactate accumulation during the time trial. IMTG did not change with treatment or exercise. PDH-E1α Ser(293) and Ser(300) phosphorylation were lower (P<0.05) before the submaximal exercise with TER than PLA with no difference after the submaximal exercise and the time trial. Before the submaximal exercise, ACC2 Ser(221) phosphorylation was higher (P<0.05) with TER than PLA. There was no difference in αAMPK Thr(172) phosphorylation between treatments. The present study suggests that beta2-agonists do not enhance 300-kcal time trial performance, but increase carbohydrate metabolism in skeletal muscles during submaximal exercise independent of AMPK and ACC phosphorylation, and that this effect diminishes as drug exposure time, exercise duration and intensity are increased.