Disruption of intraislet mechanisms could account for the impaired glucagon response to hypoglycemia in type 1 diabetes. However, in contrast to animals, there is conflicting evidence that such mechanisms operate in humans. We have used iv tolbutamide (T) (1.7 g bolus + 130 mg/h infusion) to create high portal insulin concentrations and compared this with equivalent hypoglycemia using an iv insulin infusion (I) (30 mU/m²·min). Ten normal subjects underwent two hypoglycemic clamps; mean glucose; I (53 ± 1 mg/dL); and T (53 ± 1 mg/dL) (2.9 ± 0.04 mmol/L vs. 2.9 ± 0.05 mmol/L), held for 30 min. During hypoglycemia, mean peripheral insulin levels were greater with I (59 ± 4 mU/L) than T (18 ± 3 mU/L), P < 0.001. Calculated peak portal insulin concentrations were greater during T (282 ± 28 mU/L) than I (78 ± 4 mU/L), P < 0.00005. The demonstration of a reduced glucagon response during T-induced hypoglycemia (111 ± 8 ng/L vs. 135 ± 12 ng/L, P < 0.05) with higher portal insulin concentrations suggests that intraislet mechanisms may contribute to the release of glucagon during hypoglycemia in man.