In the last decade, several secreted and cytosolic mammalian phospholipase A₂ (PLA₂) isozymes have been described. The existence of several types of PLA₂ in phagocytic cells complicates delineation of the PLA₂ responsible for release of arachidonic acid (AA) following phagocyte stimulation. Cytosolic phospholipase A₂ (cPLA₂) was shown to be activated by phosphorylation on serine residue (505) by MAP kinase (ERKs or p38) and to translocate to the membranes in response to calcium concentrations found in stimulated cells. Our studies, which were performed in differentiated cPLA₂-deficient PLB-985 cells, are the first to demonstrate the effects of complete and specific inhibition of cPLA₂ protein expression, and strongly implicate cPLA₂ as the PLA₂ isozyme responsible for the majority of pulse released arachidonic acid following stimulation of phagocytes. This model also provides the first unequivocal demonstration that arachidonic acid release mediated by cPLA₂ is an absolute requirement for superoxide generation by the NADPH oxidase. This effect was found to be highly specific since other responses such as phagocytosis or chemotaxis were found to be normal in the cPLA₂ knock out cell line. The importance of cPLA₂ in mediating a variety of functional responses in phagocytes has been demonstrated in several studies using antisense techniques to inhibit cPLA₂ expression. [³H] AA release or PGE₂ formation generated by cPLA₂ were induced with monocyte chemotactic protein -1, bacterial lipopolysaccharide, or monocyte colony stimulating factor.   However, cPLA₂ is not involved in either proliferation or differentiation processes.