In normal b-cells glucose-stimulated insulin secretion (GSIS) results from 1) closure of K_ATP channels, depolarization, Ca²⁺ influx and rise in cytosolic [Ca²⁺]_c (triggering pathway) and 2) augmentation of the action of Ca²⁺ on exocytosis (amplifying pathway). We previously reported that the amplifying pathway largely accounts for GSIS in adult b-cells lacking K_ATP channels because of an inactivation of sulfonylurea receptor 1 (Sur1KO). Here we compared [Ca²⁺]_c and IS in overnight cultured islets from 2 week-old normal and Sur1KO mice. Most experiments were done in the presence of forskolin to increase islet cAMP levels. Two week-old normal islets proved functionally mature: magnitude and biphasic kinetics of [Ca²⁺]_c and IS changes induced by glucose, and operation of the amplifying pathway were similar to adult islets. Sur1KO islets perifused with 1 mM glucose showed elevation of basal [Ca²⁺]_c and IS. Stimulation with 15 mM glucose produced a transient drop of [Ca²⁺]_c followed by an overshoot and a sustained elevation beyond initial levels, accompanied by a monophasic, 6-fold increase in IS. Glucose also increased IS when [Ca²⁺]_c was clamped by 30 mM KCl. When Sur1KO islets were cultured in 5 instead of 10 mM glucose, [Ca²⁺]_c and IS were unexpectedly low in 1 mM glucose, and increased following a biphasic time-course upon stimulation by 15 mM glucose. This K_ATP channel-independent biphasic [Ca²⁺]_c rise was attributed to a Na⁺-, Cl^-- and Na⁺ pump-independent depolarization of b-cells, leading to Ca²⁺ influx through voltage-dependent Ca channels. In conclusion, we show that acute and chronic metabolic control of [Ca²⁺]_c is possible in b-cells lacking K_ATP channels, and suggest that unidentified K channels sensitive to glucose subserve this control. GSIS in Sur1KO islets thus involves the expected operation of the amplifying pathway and unexpected changes in the triggering signal.