The cytoplasmic Ca2+ concentration ([Ca2+]i) and insulin release were measured simultaneously in mouse pancreatic islets cultured overnight. [Ca2+]i was 105 nM and insulin release 3 pmol.g-1.s-1 at 3 mM glucose. An increase to 7 mM glucose reduced [Ca2+]i transiently, whereas insulin release doubled and was pulsatile with a frequency of 0.47 min-1. [Ca2+]i oscillations with similar frequency appeared at 11 mM glucose associated with increased amplitude of the insulin oscillations, raising the secretory rate 10-fold. In the presence of 16 and 20 mM glucose [Ca2+]i was > 300 nM and showed no oscillations apart from two islets, which demonstrated [Ca2+]i oscillations with small amplitude at 16 mM glucose. Insulin release with maintained frequency increased by 46 and 31%, respectively. When the glucose concentration was increased from 3 to 11 mM, [Ca2+]i decreased with a nadir that appeared significantly earlier than when the glucose concentration was raised from 3 to 7 mM. Glucose-induced insulin release from the isolated islet is pulsatile both at stable and oscillatory [Ca2+]i, with changes in secretory rate caused by the sugar also when [Ca2+]i is unchanged.