The ability of chronic endogenous hyperinsulinemia to induce a resistance to insulin action on protein and glucose metabolism was studied in 10 subjects affected by a benign (functioning) insulinoma and 18 healthy subjects by means of infusions of [l-14C]leucine and [3-3H] glucose. The insulinoma subjects were divided into two groups with moderate (139 ± 12 pmol/1) (n = 5) and marked (438 ± 42 pmol/1) (n = 5) hyperinsulinemia and were studied during a euglycemic dextrose infusion. Control subjects were studied postabsorptively and during a low-dose (0.3 mU · kg−1·min−1) (n = 3) and a high-dose (1 mU · kg−1 · min−1) (n = 15) euglycemic insulin clamp to match peripheral insulin concentrations with those of insulinoma subjects. In insulinoma subjects there was no correlation among plasma insulin concentration and leucine concentration (r = 0.05), endogenous leucine flux (r = 0.44), hepatic glucose production (r = 0.47), and glucose uptake (r = 0.05). Insulinoma subjects with marked hyperinsulinemia demonstrated a defective suppression of leucine concentrations (100 ± 11 vs. 65 ± 5 μmol/l,P < 0.01), endogenous leucine flux (50.1 ± 6.3 vs. 27.1 ± 0.9 μmol · m−2 · min−1, P < 0.01), and hepatic glucose production (5.4 ± 2.0 vs. 0.6 ± 0.6 ± mol · kg−1 · min−1, P < 0.05), and a defective stimulation of glucose uptake (13.5 ± 1.6 vs. 41.1 ± 2.8 ± mol · kg−1 · min−1, P <0.001) with respect to normal subjects at a comparable degree of hyperinsulinemia. Our results demonstrate that chronic endogenous hyperinsulinemia causes a defect in insulin action that is generalized to both protein metabolism and hepatic and peripheral glucose metabolism.