Intensifying pharmacological therapy in patients with type 2 diabetes increases the risk of hypoglycemia and often requires the simultaneous use of more than one agent. Combining insulin and sulfonylurea is an effective and frequently used therapy in such patients. However, sulfonylurea derivatives have been shown to affect the release of glucagon, indicating a possible effect of such therapy on hormonal counterregulation to hypoglycemia. Thirteen patients receiving combined therapy were studied on two occasions: 1) after a wash-out period of glibenclamide (-GLIB), and 2) after resuming combined treatment for 6 months (+GLIB). We performed nonstep-wise, hyperinsulinemic hypoglycemic clamps using a constant i.v. insulin infusion and clamping blood glucose at 2.7 mmol/L (48 mg/dL) for 60 min. C Peptide levels were significantly higher during + GLIB, but no significant differences were seen in peripheral plasma insulin levels (+GLIB mean +/- SD, 70 +/- 17 mU/L vs. -GLIB, 75 +/- 14; P = 0.26). Epinephrine responses were similar in the two tests, but when glibenclamide was present the glucagon response was smaller, both the peak value (P = 0.016) and the incremental area under the curve (P = 0.011) as well as the total area under the curve (P = 0.016). These results suggest that intraislet insulin secretion is of importance for the alpha-cell responsiveness to hypoglycemia in these patients.