Phosphatidylinositol-3-phosphate (PtdIns(3)P) is a key player in early endosomal trafficking and is mainly produced by class III PI3-kinase. In neurosecretory cells, class II PI3-kinase C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3-kinase C2alpha knock down and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3-kinase C2alpha knock down and expression of PI3-kinase C2alpha catalytically-inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly-expressed PI3-kinase C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3-kinase C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3 kinase family is regulated by Ca(2+) in vitro and in living neurosecretory cells.