Mutations in the high-affinity sulfonylurea receptor (SUR)-1 cause one of the severe recessively inherited diffuse forms of congenital hyperinsulinism or, when associated with loss of heterozygosity, focal adenomatosis. We hypothesized that SUR1 mutations would render the β-cell insensitive to sulfonylureas and to glucose. Stimulated insulin responses were compared among eight patients with diffuse hyperinsulinism (two mutations), six carrier parents, and ten normal adults. In the patients with diffuse hyperinsulinism,the acute insulin response to intravenous tolbutamide was absent and did not overlap with the responses seen in either adult group. There was positive,albeit significantly blunted, acute insulin response to intravenous dextrose in the patients with diffuse hyperinsulinism. Graded infusions of glucose, to raise and then lower plasma glucose concentrations over 4 h, caused similar rises in blood glucose but lower peak insulin levels in the hyperinsulinemic patients. Loss of acute insulin response to tolbutamide can identify children with diffuse SUR1 defects. The greater response to glucose than to tolbutamide indicates that ATP-sensitive potassium (K_ATP) channel-independent pathways are involved in glucose-mediated insulin release in patients with diffuse SUR1 defects. The diminished glucose responsiveness suggests that SUR1 mutations and lack of K_ATP channel activity may contribute to the late development of diabetes in patients with hyperinsulinism independently of subtotal pancreatectomy.