The effects of physiological hyperinsulinemia (approximately 75 mU/l) on glucose storage, oxidation, and glycolysis in skeletal muscle were assessed with euglycemic clamps performed in seven healthy volunteers, in conjunction with leg balance for glucose, lactate, alanine, O2, and CO2. Infusion of insulin increased leg glucose uptake, storage, and oxidation but did not alter net release of lactate and alanine. The respiratory quotient (RQ) across the leg increased from a basal value of 0.74 +/- 0.02 to 0.99 +/- 0.02 during hyperinsulinemia. Under conditions of insulin stimulation, 49 +/- 5% of leg glucose uptake was stored, 37 +/- 4% was oxidized, and 14 +/- 2% was released as lactate and alanine. We conclude that during physiological hyperinsulinemia and euglycemia 1) skeletal muscle lipid oxidation is nearly entirely suppressed and glucose becomes the primary oxidative substrate of muscle, 2) glucose storage and oxidation are the major pathways of skeletal muscle glucose metabolism and are quantitatively similar at physiological insulin levels, and 3) the majority of insulin-stimulated glycolysis is oxidized, with only a small portion released as lactate or alanine.