An increase in cytoplasmic Ca²⁺ in β-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) directly regulate secretion remains disputed. This question was addressed by investigating the temporal and quantitative relationships between [Ca²⁺]_i and insulin secretion. Both events were measured simultaneously in single mouse islets loaded with fura-PE3 and perifused with a medium containing diazoxide (to prevent any effect of glucose on the membrane potential) and either 4.8 or 30 mmol/l K⁺. Continuous depolarization with 30 mmol/l K+ in the presence of 15 mmol/l glucose induced a sustained rise in [Ca²⁺]_i and insulin release. No oscillations of secretion were detected even after mathematical analysis of the data (pulse, spectral and sample distribution analysis). In contrast, alternating between 30 and 4.8 mmol/l K⁺ (1 min/2 min or 2.5 min/5 min) triggered synchronous [Ca²⁺]_i and insulin oscillations of regular amplitude in each islet. A good correlation was found between [Ca²⁺]_i and insulin secretion, and it was independent of the presence or absence of oscillations. This quantitative correlation between [Ca²⁺]_i and insulin secretion was confirmed by experiments in which extracellular Ca²⁺ was increased or decreased (0.1–2.5 mmol/l) stepwise in the presence of 30 mmol/l K⁺. This resulted in parallel stepwise increases or decreases in [Ca²⁺]_i and insulin secretion. However, while the successive [Ca²⁺]_i levels were unaffected by glucose, each plateau of secretion was much higher in 20 than in 3 mmol/l glucose. In conclusion, in our preparation of normal mouse islets, insulin secretion oscillates only when [Ca²⁺]_i oscillates in β-cells. This close temporal relationship between insulin secretion and [Ca²⁺]_i changes attests of the regulatory role of Ca²⁺. There also exists a quantitative relationship that is markedly influenced by the concentration of glucose.