The role of pyruvate dehydrogenase complex (PDC) in insulin-stimulated glycogen replenishment the day after exercise, and its molecular control, has not been examined. This study investigated the effect of acute exercise on basal and insulin-stimulated PDC activity (the rate-limiting step in glucose oxidation), glycogen synthesis and the expression of metabolic genes and transcription factors associated with changes in PDC activation and glucose metabolism. Eight healthy men (age 24 +/- 2 years, body mass 79 +/- 4 kg) underwent a euglycaemic, hyperinsulinaemic clamp 22 h after 90 min of one-legged cycling at 60% maximal oxygen consumption. Skeletal muscle glycogen content was similar in the exercised (EX) and non-exercised leg (CON) preclamp (471 +/- 30 versus 463 +/- 50 mmol (kg dry matter)(1), respectively) but increased during the clamp in EX to 527 +/- 20 mmol (kg dry matter)(1), such that it was 17% greater than in CON (449 +/- 35 mmol (kg dry matter)(1), P < 0.05). This increase in insulin-mediated glycogen storage was independent of insulin-stimulated Akt serine(473) phosphorylation and activation of PDC. Prior exercise did not modulate the mRNA expression and protein content of pyruvate dehydrogenase kinase 4 (PDK4) in skeletal muscle, but was associated with increased hexokinase II mRNA expression and protein content and upregulation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC1alpha) and PPARdelta gene expression. Collectively, these findings suggest that prior exercise does not alter basal and insulin-stimulated PDC activation and the protein content of PDK4 the following day, but is associated with increased capacity (through upregulation of hexokinase II content) of muscle to phosphorylate and divert glucose towards glycogen storage.