Arachidonic acid (AA) can trigger activation of the phagocyte NADPH oxidase in a cell-free assay. However, a role for AA in activation of the oxidase in intact cells has not been established, nor has the AA generating enzyme critical to this process been identified. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A₂(cPLA₂) antisense mRNA and stable clones were selected that lack detectable cPLA₂. cPLA₂-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D₃, indicating that cPLA₂ is not involved in the differentiation process. Neither cPLA₂ nor stimulated [³H]AA release were detectable in differentiated cPLA₂-deficient PLB-985 cells, demonstrating that cPLA₂ is the major type of PLA₂ activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA₂-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but the addition of exogenous AA fully restores oxidase activity. This establishes an essential requirement of cPLA₂-generated AA for activation of phagocyte NADPH oxidase.