To study whether impaired activation of muscle glycogen synthase represents an early defect in the pathogenesis of insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM), we quantitated rates of nonoxidative glucose metabolism and measured activities of glycogen synthase and phosphorylase and concentrations of free glucose and glucose-6-phosphate in muscle biopsies, obtained before and after a euglycemic insulin clamp, in 16 NIDDM patients, 18 first-degree relatives of NIDDM patients, and 16 nondiabetic control subjects. Insulin-stimulated glucose storage (20.1 +/- 1.5 and 11.6 +/- 1.7 vs. 27.9 +/- 1.7 mumol.kg-1 lean body mass [LBM].min-1, P less than 0.01-0.001 [3.6 +/- 0.3 and 2.1 +/- 0.3 vs. 5.0 +/- 0.3 mg.kg-1 LBM.min-1] and glycogen synthase activity, measured at 0.1 mM glucose-6-phosphate concentration (11.3 +/- 1.3 and 11.6 +/- 1.3 vs. 18.3 +/- 2.0 nmol.min-1.mg-1 protein, P less than 0.01), were impaired in relatives and diabetic subjects compared with control subjects. Glycogen synthase activity correlated with the rate of glucose storage (r = 0.53, P less than 0.001). Glycogen phosphorylase fractional activity did not differ among the groups. Apart from increased intramuscular basal glucose concentrations in NIDDM patients, no consistent differences were observed in free glucose and glucose-6-phosphate concentrations between the groups. We conclude that impaired activation of muscle glycogen synthase by insulin is observed in patients with a genetic risk of developing NIDDM and may represent an early defect in the pathogenesis of NIDDM.