Phosphatidylinositol-4, 5-bisphosphate [PI(4,5)P(2)] has been implicated in the priming of large dense-core vesicles in many secretory cells; however, its role in the Ca(2+)-dependent secretory activity in pituitary cells remains elusive. We assessed the effect of elevated intracellular PI(4,5)P(2) on the kinetics of Ca(2+)-dependent exocytosis, using a whole-cell patch-clamp technique in wild-type mouse melanotrophs from fresh pituitary tissue slices. We found that 1 micromol/L PI(4,5)P(2) significantly increased Ca(2+)-dependent exocytosis of vesicles that need to go through ATP-dependent reactions; however, the exocytosis of release-ready vesicles (ATP-independent release) and voltage-activated Ca(2+) currents remained unaffected. We suggest that PI(4,5)P(2) increases the size of the readily releasable vesicle pool by regulating the effectiveness of vesicular mobilization and fusion in an ATP-dependent manner.