Inhibition of ATP-sensitive K⁺ (K_ATP) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic β-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1^−/−) in β-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1^−/− mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-1 (GLP-1) on IR and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) of freshly isolated or cultured islets of SUR1^−/− mice and B6D2F1 controls (SUR1^+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1^−/− islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1^−/− islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca²⁺ stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca²⁺]_i at 0.1–1 μM or biphasic changes (an initial sharp increase in [Ca²⁺]_i followed by a sustained phase of low [Ca²⁺]_i) at 1–100 μM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the β-cells and markedly elevates basal [Ca²⁺]_i. Elevated [Ca²⁺]_i in turn sensitizes the β-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca²⁺]_i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia.