This study examined the effect of pre-exercise carbohydrate (CHO) ingestion on pyruvate dehydrogenase complex (PDC) activation, acetyl group availability and substrate level phosphorylation (glycogenolysis and phosphocreatine (PCr) hydrolysis) in human skeletal muscle during the transition from rest to steady-state exercise. Seven male subjects performed two 10 min treadmill runs at 70 % maximum oxygen uptake (VO2,max), 1 week apart. Each subject ingested 8 ml (kg body mass (BM))-1 of either a placebo solution (CON trial) or a 5.5 % CHO solution (CHO trial) 10 min before each run. Muscle biopsy samples were obtained from the vastus lateralis at rest and immediately after each trial. Muscle PDC activity was higher at the end of exercise in the CHO trial compared with the CON trial (1.78+/-0.18 and 1.27+/-0.16 mmol min(-1) (kg wet matter (WM))(-1), respectively; P 0.05) and this was accompanied by lower acetylcarnitine (7.1+/-1.2 and 9.1+/-1.1 mmol kg(-1) (dry matter (DM))(-1) in CHO and CON, respectively; P<0.05) and citrate concentrations (0.73+/-0.05 and 0.91+/-0.10 mmol (kg DM)(-1) in CHO and CON, respectively; P<0.05). No difference was observed between trials in the rates of muscle glycogen and PCr breakdown and lactate accumulation. This is the first study to demonstrate that CHO ingestion prior to exercise augments the exercise-induced activation of muscle PDC and reduces acetylcarnitine accumulation during the transition from rest to steady-state exercise. However, those changes did not affect the contribution of substrate level phosphorylation to ATP resynthesis.