Rapid and sustained stimulation of β-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each β-cell rather than heterogeneity in the time-course of the response between β-cells or islets. There is no evidence indicating that biphasic secretion can be attributed to an intrinsically biphasic metabolic signal. In contrast, the biphasic rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) induced by glucose is important to shape the two phases of secretion. The first phase requires a rapid and marked elevation of [Ca²⁺]_i and corresponds to the release of insulin granules from a limited pool. The magnitude of the second phase is determined by the elevation of [Ca²⁺]_i, but its development requires production of another signal. This signal corresponds to the amplifying action of glucose and may serve to replenish the pool of granules that are releasable at the prevailing [Ca²⁺]_i. The species characteristics of biphasic insulin secretion and its perturbations in pathological situations are discussed.