While lowering muscle glycogen availability to an extent that would reduce muscle pyruvate formation during intense exercise, we investigated the importance of muscle pyruvate availability to pyruvate dehydrogenase complex (PDC) activation during intense exercise in human skeletal muscle. The present study demonstrated that regardless of whether pre-exercise muscle glycogen content was at a habitual resting concentration (412 +/- 30 mmol (kg dry muscle)(-1)) or depleted (60 +/- 3 mmol (kg dry muscle)(-1)), the increase in PDC activation from its resting value (5.46 +/- 0.96 and 3.67 +/- 0.34 nmol acetyl-CoA min(-1) (mg protein)(-1), respectively) during 10 min of exercise at 75% of the maximum rate of oxygen consumption (VO2max) (delta12.82 +/- 1.72 and delta13.24 +/- 1.42 nmol acetyl-CoA min(-1) (mg protein)(-1), respectively) was the same, despite pyruvate accumulation during exercise being 3-fold lower in the glycogen depleted state (delta0.34 +/- 0.04 and delta0.11 +/- 0.06 mmol (kg dry muscle)(-1), P < 0.001). However, as a result of the reduction in pyruvate availability, calculated flux through the PDC reaction was at least 2-fold lower in the glycogen depleted state compared with normal (21.81 +/- 2.62 and 9.41 +/- 0.63 nmol acetyl-CoA min(-1) (mg protein)(-1), respectively; P < 0.001). It is therefore pertinent to conclude that whilst muscle pyruvate availability appears to be important to the rate of flux through the PDC reaction during in vivo contraction, it is not of primary importance to the control of PDC activation under these conditions, which is probably principally regulated by muscle calcium availability. The proposed central role of pyruvate in muscle PDC activation during in vivo contraction may therefore have been over stated.