Chemically elicited guinea pig peritoneal exudate macrophages respond by Superoxide (O 2 −) production to a large number of unrelated stimulants. It has been found that 8 out of 10 stimulants also induce arachidonic acid (20:4) liberation and thromboxane synthesis. The elicitation of O 2 − production by most stimulants was reduced or totally suppressed by three procedures that inhibit the activity of endogenous phospholipases: the use of drug p-bromophenacyl bromide, elevation of the cellular cyclic AMP level, and the removal of extracellular Ca 2+. O 2 − production in response to concanavalin A, wheat germ agglutinin, and fMet-Leu-Phe were exquisitely sensitive to inhibition of phospholipase activity. Exogenously applied 20:4 as well as other unsaturated fatty acids (linolenic, linoleic, and oleic) induced massive and instantaneous O 2 − production in a dose-dependent manner. Saturated fatty acids (stearic) and methyl esters of unsaturated acids were inactive. Lysophosphoglycerides were also inactive. Incubation of macrophages with inhibitors of cyclooxygenase or lipoxygenase did not prevent the elicitation of O 2 − production by stimulants or fatty acids. On the contrary, O 2 − formation was enhanced by indomethacin and indomethacin by itself was capable of evoking O 2 − generation. Treatment of 20:4 with soybean lipoxygenase did not abolish its capacity to induce O 2 − production; native and lipoxygenasetreated 20:4 exhibited similar dose-response ratios. Purified 15-hydroxyeicosatetraenoic acid also elicited O 2 − production by macrophages with a potency comparable to but not exceeding that of 20:4. Equimolar amounts of prostaglandin E 2 were inactive. These findings suggest that liberation of unsaturated fatty acid (principally, 20:4) from membrane phospholipids, as a consequence of phospholipase activation, is a necessary step in the elicitation of an oxidative burst in macrophages. O 2 − generation is stimulated by unesterified 20:4 and, possibly, by certain metabolites of 20:4. It appears that the lipoxygenase pathway may generate metabolites with stimulating capacity while the cyclooxygenase pathway is abortive.