We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A 2 (sPLA 2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA 2-IIA,-V, and-IID), but not heparin-nonbinding (sPLA 2-IIC), enzymes released more granule-associated markers (β-hexosaminidase and histamine) than mock-or cytosolic PLA 2 α (cPLA 2 α)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA 2-IIA and-V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA 2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA 2 s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA 2-IIA,-IID, and-V in the regulation of degranulation, only sPLA 2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD 2 production, which reached a level comparable to that elicited by cPLA 2 α. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.