Glucose-activated beta-cell insulin secretion depends upon elevation of intracellular calcium concentration, [Ca2+]i, which is thought to arise from Ca2+ influx through voltage-dependent calcium channels. Using fura-2-loaded mouse islets, we demonstrate, in fact, that the major component of the glucose-activated [Ca2+]i rise represents voltage-dependent intracellular Ca2+ release. Furthermore, the Ca2+ release pool possesses a novel pharmacology in that it is caffeine-sensitive but ryanodine-insensitive. In the absence of external Ca2+, glucose still caused intracellular Ca2+ release, an effect blockable by tetrodotoxin. However, depolarization of the islet with KCl in low Ca(2+)-containing solutions induced intracellular Ca2+ release, which was resistant to tetrodotoxin. We conclude that glucose release of intracellular Ca2+ is dependent upon depolarization alone, possibly through increasing inositol 1,4,5-trisphosphate production.