The mechanisms underlying biphasic insulin secretion have not been completely elucidated. We compared the pattern of plasma insulin changes during hyperglycemic clamps in mice to that of glucose-induced insulin secretion and cytosolic calcium concentration ([Ca²⁺]_c) changes in perifused mouse islets. Anesthetized mice were infused with glucose to clamp blood glucose at 8.5 (baseline), 11.1, 16.7, or 30 mmol/l. A first-phase insulin response consistently peaked at 1 min, and a slowly ascending second phase occurred at 16.7 and 30 mmol/l glucose. Glucose-induced insulin secretion in vivo is thus biphasic, with a similarly increasing second phase in the mouse as in humans. In vitro, square-wave stimulation from a baseline of 3 mmol/l glucose induced similar biphasic insulin secretion and [Ca²⁺]_c increases, with sustained and flat second phases. The glucose dependency (3–30 mmol/l) of both changes was sigmoidal with, however, a shift to the right of the relation for insulin secretion compared with that for [Ca²⁺]_c. The maximum [Ca²⁺]_c increase was achieved by glucose concentrations, causing half-maximum insulin secretion. Because this was true for both phases, we propose that contrary to current concepts, amplifying signals are also implicated in first-phase glucose-induced insulin secretion. To mimic in vivo conditions, islets were stimulated with high glucose after being initially perifused with 8.5 instead of 3.0 mmol/l glucose. First-phase insulin secretion induced by glucose at 11.1, 16.7, and 30 mmol/l was decreased by ∼ 50%, an inhibition that could not be explained by commensurate decreases in [Ca²⁺]_c or in the pool of readily releasable granules. Also unexpected was the gradually ascending pattern of the second phase, now similar to that in vivo. These observations indicated that variations in prestimulatory glucose can secondarily affect the magnitude and pattern of subsequent glucose-induced insulin secretion.