Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.