Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2α 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 PI3K-C2α knockdown 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 PI3K-C2α knockdown and expression of PI3K-C2α catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca2+. We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2α is indeed regulated by Ca2+. We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2α occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca2+in vitro and in living neurosecretory cells.