Phosphatidic acid (PA), the product of phospholipase D (PLD) metabolism, is not only an important second messenger in neutrophil signal transduction but PA generation increases membrane fusogenicity, Following neutrophil stimulation, PA formation can be detected in azurophil, specific, and plasma membrane vesicle subcellular fractions, suggesting a potential role for PA formation in granule-plasma membrane fusion, Neutrophil stimulation also activates phospholipase A(2) (PLA(2)) and the release of arachidonic acid. In vitro fusion of plasma membrane vesicles and specific granules with complex liposomes were dependent on PLA(2) (<10 M Ca2+) while the presence of PA in the liposomes augmented the effects of PLA(2). Azurophil granules were extremely resistant to fusion (no fusion at 12 mM Ca2+ even in the presence of PLA(2)). However, in the presence of both PA and PLA(2) fusion could be detected at <5 M Ca2+, suggesting a direct role for phospholipid metabolism in neutrophil degranulation.