Insulin resistance is accentuated during periods of poor metabolic control in human non-insulin-dependent diabetes mellitus. The role of hyperglycemia in this suppression of insulin action is not clear. If glucose impairs insulin action, then the effect should be reproducible in vivo in tissues of normal intact rats. To test this possibility, normal rats were continuously administered 50% glucose in water (60-66 mg.kg-1.min-1) via an indwelling jugular catheter. After 72 h, these animals were hyperglycemic, hyperinsulinemic, and glucosuric compared with control rats infused for 72 h with normal saline (P less than 0.01). Basal glucose uptake in vivo was greater in muscle of glucose-infused rats. Insulin-stimulated glucose uptake in vivo and in vitro (by perfused hindquarters and isolated adipocytes) were suppressed in the glucose-infused group (P less than 0.01). Glycogen synthase activity was reduced 40% in extracts of muscle and adipose tissue of hyperglycemic rats. Basal and isoproterenol-stimulated lipolysis were increased, whereas insulin suppression of lipolysis was blunted in adipocytes from glucose-infused animals (P less than 0.01). Glucose infusion did not alter insulin binding by isolated adipocytes or solubilized skeletal muscle insulin receptors. These results suggest that a 72-h in vivo glucose infusion impaired insulin action in muscle and adipose tissue of normal rats by inducing postbinding defects similar to those observed in human diabetes mellitus during intervals of deteriorated metabolic control.