Congenital hyperinsulinism (CHI) is related to two main histological pancreas anomalies: focal adenomatous hyperplasia and diffuse β-cell hypersecretion. Pharmacological tests to measure acute insulin responses (AIR) to peripheral iv injections of glucose, calcium, and tolbutamide have been reported as potential means to distinguish between these histological forms. In patients with defects in ATP-sensitive potassium channels, tolbutamide will fail to induce insulin release in affected portions of the pancreas, whereas calcium gluconate will enhance insulin release through spontaneously active voltage-gated Ca²⁺ channels. Consequently, in focal CHI patients, calcium should promote AIRs from the lesion, whereas tolbutamide should act to promote insulin secretion from the healthy region of the pancreas (outside the focal hyperplasia). We therefore studied AIRs to calcium and tolbutamide stimulation tests in 16 children with focal (n = 9) or diffuse (n = 7) CHI before pancreatic surgery. We found hypervariable AIRs to glucose and calcium stimulation in both focal and diffuse CHI patients. AIRs to tolbutamide stimulation were found modest in focal CHI patients, which might account for β-cell quiescence in the healthy portion of the pancreas of these patients. We conclude that AIRs to calcium and tolbutamide stimulation tests are not sufficient to differentiate the focal from the diffuse CHI patients.