Insulin receptor function, glycogen synthase activity, and activation by phosphatases were studied in biopsies of human skeletal muscle under conditions of hyperglycemia and/or hyperinsulinemia for 150 minutes. Twenty-one healthy volunteers underwent either (A) a hyperinsulinemic, euglycemic clamp (serum insulin, 160.0 ± 7.7 mU/L; plasma glucose, 4.9 ± 0.1 mmol/L; n = 9), (B) a hyperglycemic clamp during normoinsulinemia (serum insulin, 18.1 ± 3.3 mU/L; plasma glucose, 12.9 ± 0.2 mmol/L; n = 6), or (C) a combined hyperinsulinemic, hyperglycemic clamp (serum insulin, 158.3 ± 15.0 mU/L; plasma glucose, 11.4 ± 0.8 mmol/L; n = 6). During all studies, the endogenous insulin secretion was inhibited with somatostatin. Insulin binding and kinase activity of insulin receptors solubilized from vastus lateralis muscle biopsies were unaffected by hyperglycemia and/or hyperinsulinemia. Hyperinsulinemia activated the muscle glycogen synthase with a decrease in the half-maximal activation constant (A_0.5) for glucose-6-phosphate (G6P) from 0.53 ± 0.04 to 0.21 ± 0.02 mmol/L (study A, P < .02) and from 0.53 ± 0.06 to 0.19 ± 0.05 mmol/L (study C, P < .03). In addition, the rate of glycogen synthase activation by phosphatases increased from 0.078 ± 0.017 to 0.134 ± 0.029 U/min/mg protein (study A, P < .03) and from 0.082 ± 0.013 to 0.145 ± 0.033 U/min/mg protein (study C, P = .05). Hyperglycemia during normoinsulinemia did not affect A_0.5 or phosphatase activity. In conclusion, (1) hyperinsulinemia for 2 1 2 hours increases glycogen synthase activity and activation by phosphatases independently on the glycemia; and (2) insulin receptor binding and basal and insulin-stimulated receptor kinase activity are not modified during short-term hyperinsulinemia and/or hyperglycemia.