The roles of pyruvate dehydrogenase (PDH), glycogen phosphorylase (Phos), and their regulators in lactate (Lac(-)) metabolism were examined during incremental exercise after ingestion of 0.3 g/kg of either NH(4)Cl [metabolic acidosis (ACID)] or CaCO(3) [control (CON)]. Subjects were studied at rest, at rest postingestion, and during continuous steady-state cycling at three stages (15 min each): 30, 60, and 75% of maximal oxygen uptake. Radial artery and femoral venous blood samples, leg blood flow, and biopsies of the vastus lateralis were obtained during each power output. ACID resulted in significantly lower intramuscular concentration of [Lac(-)] (ACID 40.8 vs. CON 56.9 mmol/kg dry wt), arterial whole blood [Lac(-)] (ACID 4.7 vs. CON 6.5 mmol/l), and leg Lac(-) efflux (ACID 3.05 vs. CON 6.98 mmol. l(-1). min(-1)). The reduced intramuscular [Lac(-)] resulted from decreases in pyruvate production due to inhibition of glycogenolysis, at the level of Phos a, and phosphofructokinase, together with an increase in the amount of pyruvate oxidized relative to the total produced. The reduction in Phos a activity was mediated through decreases in transformation, decreases in free inorganic phosphate concentration, and decreases in the posttransformational allosteric regulator free AMP. Reduced PDH activity occurred with ACID and may have resulted from alterations in the concentrations of acetyl-CoA, free ADP, pyruvate, NADH, and H(+), leading to greater relative activity of the kinase. The results demonstrate that imposed metabolic acidosis in skeletal muscle results in decreased Lac(-) production due to inhibition of glycogenolysis at the level of Phos and increased pyruvate oxidation at PDH.