Capacitance measurements were applied to mouse pancreatic β-cells to elucidate the cellular mechanisms underlying biphasic insulin secretion. We report here that only <50 of the β-cell’s >10,000 granules are immediately available for release. The releasable granules tightly associate with the voltage-gated α_1C Ca²⁺ channels, and it is proposed that the release of these granules accounts for first-phase insulin secretion. Subsequent replenishment of the releasable pool by priming of previously nonreleasable granules is required for second-phase insulin secretion. The latter reaction depends on intragranular acidification due to the concerted action of granular bafilomycin-sensitive v-type H⁺-ATPase and 4,4-diisothiocyanostilbene-2,2-disulfonate–blockable ClC-3 Cl⁻ channels. Lowering the cytoplasmic ATP/ADP ratio prevents granule acidification, granule priming, and refilling of the releasable pool. The latter finding provides an explanation to the transient nature of insulin secretion elicited by, for example, high extracellular K⁺ in the absence of metabolizable fuels.