Hyperinsulinism of infancy (HI) is a congenital defect in the regulated release of insulin from pancreatic β-cells. Here we describe stimulus-secretion coupling mechanisms in β-cells and intact islets of Langerhans isolated from three patients with a novel SUR1 gene defect. 2154+3 A to G SUR1 (GenBank accession number L78207) is the first report of familial HI among nonconsanguineous Caucasians identified in the U.K. Using patch-clamp methodologies, we have shown that this mutation is associated with both a decrease in the number of operational ATP-sensitive K⁺ channels (K_ATP channels) in β-cells and impaired ADP-dependent regulation. There were no apparent defects in the regulation of Ca²⁺- and voltage-gated K⁺ channels or delayed rectifier K⁺ channels. Intact HI β-cells were spontaneously electrically active and generating Ca²⁺ action currents that were largely insensitive to diazoxide and somatostatin. As a consequence, when intact HI islets were challenged with glucose and tolbutamide, there was no rise in intracellular free calcium ion concentration ([Ca²⁺]_i)over basal values. Capacitance measurements used to monitor exocytosis in control and HI β-cells revealed that there were no defects in Ca²⁺-dependent exocytotic events. Finally, insulin release studies documented that whereas tolbutamide failed to cause insulin secretion as a consequence of impaired [Ca²⁺]_i signaling, glucose readily promoted insulin release. Glucose was also found to augment the actions of protein kinase C— and protein kinase A—dependent agonists in the absence of extracellular Ca²⁺. These findings document the relationship between SUR1 gene defects and insulin secretion in vivo and in vitro and describe for the first time K_ATPchannel—independent pathways of regulated insulin secretion in diseased human β-cells.