T he mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.