Objectives: To identify infants with hyperinsulinism caused by defects of the β-cell adenosine triphosphate-dependent potassium channel complex and to distinguish focal and diffuse forms of hyperinsulinism caused by these mutations. Study design: The acute insulin response to intravenous calcium stimulation (CaAIR) was determined in 9 patients <20 years with diffuse hyperinsulinism caused by defective β-cell sulfonylurea receptor (SUR1 ^–/–), 3 patients with focal congenital hyperinsulinism (6 weeks to 18 months), a 10-year-old with insulinoma, 5 with hyperinsulinism/hyperammonemia syndrome caused by defective glutamate dehydrogenase (6 months to 28 years), 4 SUR1 ^+/– heterozygotes with no symptoms, and 9 normal adults. Three infants with congenital focal disease, 1 with diffuse hyperinsulinism, and the child with insulinoma underwent selective pancreatic intra-arterial calcium stimulation with hepatic venous sampling. Results: Children with diffuse SUR1 ^–/– disease and infants with congenital focal hyperinsulinism responded to CaAIR, whereas the normal control group, patients with hyperinsulinism/hyperammonemia syndrome, and SUR1 ^+/– carriers did not. Selective arterial calcium stimulation of the pancreas with hepatic venous sampling revealed selective, significant step-ups in insulin secretion that correlated anatomically with the location of solitary lesions confirmed surgically in 2 of 3 infants with congenital focal disease and in the child with insulinoma. Selective arterial calcium stimulation of the pancreas with hepatic venous sampling demonstrated markedly elevated baseline insulin levels throughout the pancreas of the infant with diffuse hyperinsulinism. Conclusions: The intravenous CaAIR is a safe and simple test for identifying infants with diffuse SUR1 ^–/– hyperinsulinism or with focal congenital hyperinsulinism. Preoperative selective arterial calcium stimulation of the pancreas with hepatic venous sampling can localize focal lesions causing hyperinsulinism in children. The combination of these calcium stimulation tests may help distinguish focal lesions suitable for cure by local surgical resection. (J Pediatr 2000;137:239-46)