Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A₂ (cPLA₂) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca²⁺-dependent exocytosis in single mouse pancreatic -cells. cPLA₂ dose dependently (EC₅₀ = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca²⁺ current or cytoplasmic Ca²⁺ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70–80 to 280–300. cPLA₂-stimulated exocytosis was antagonized by the specific cPLA₂ inhibitor AACOCF₃. Ca²⁺-evoked exocytosis was reduced by 40% in cells treated with AACOCF₃ or an antisense oligonucleotide against cPLA₂. The action of cPLA₂ was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl^- uptake through ClC-3 Cl^- channels. Accordingly, the stimulatory action of cPLA₂ was inhibited by the Cl^- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl^- channel antisense oligonucleotides. We propose that cPLA₂ has an important role in controlling the rate of exocytosis in -cells. This effect of cPLA₂ reflects an enhanced transgranular Cl^- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.