Despite the effects of hyperinsulinemia and hyperglycemia, 2 factors known to inhibit endogenous glucose production (EGP) in nondiabetic subjects, increased EGP is a consistent feature of type 2 diabetes. Recent studies have suggested that increased glucose-6-phosphatase (G6Pase) and/or decreased glucokinase (GK) may explain the increase in EGP. However, no studies to date have clearly established this relationship in type 2 diabetes. The present studies were designed to determine rates of EGP and the activities of G6Pase and GK in obese patients scheduled for gastric bypass surgery. The study group consisted of 14 obese nondiabetic subjects and 13 patients with type 2 diabetes (BMI 53.7 +/- 2.4 vs. 50.1 +/- 1.6 kg/m2). Rates of EGP were determined after an overnight fast with a 4-h infusion of [6,6]-D-glucose, and they were significantly higher in the type 2 diabetic patients (85.9 +/- 10.0 vs. 137.8 +/- 14.4 mg x m(-2) x min(-1), P < 0.001) despite greater plasma glucose (5.1 +/- 0.1 vs. 12.0 +/- 1.1 mmol/l) and similar insulin concentrations (130.8 +/- 19.8 vs. 112.8 +/- 16.2 pmol/l, NS). Moreover, resistance to insulin-induced suppression of EGP was observed in the patients with type 2 diabetes when insulin concentrations were increased from approximately 120 to 180 pmol/l. Hepatic G6Pase activity determined from freshly isolated microsomes was significantly increased in the type 2 diabetic patients compared with the obese control subjects (0.16 +/- 0.02 vs. 0.09 +/- 0.01 micromol x min(-1) x mg(-1) protein, P < 0.02), whereas levels of GK were decreased (1.20 +/- 0.16 vs. 2.01 +/- 0.01 micromol x min(-1) x mg(-1) protein, P < 0.01). Net flux through G6Pase was significantly increased in type 2 diabetic patients (P < 0.01). We conclude that increased EGP is mediated in part by increased G6Pase flux in type 2 diabetes.