ATP-dependent priming of the secretory granules precedes Ca²⁺-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H⁺-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl⁻ uptake through granular ClC-3 Cl⁻ channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl⁻ fluxes, such as 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca²⁺-dependent exocytosis that is also likely to be operational in other secretory cell types.