Although tumor necrosis factor-alpha (TNF-alpha) has been shown to induce marked changes in the physiology/pathophysiology of cells, little is known about the effects of this cytokine on cellular lipid metabolism. In this study we examined the effects of TNF-alpha on the metabolism of eicosatetraenoic acid (arachidonic acid, (20:4(n-6)) in human neutrophils. Pretreatment of neutrophils with TNF-alpha caused a rapid increase in the incorporation of [1-14C]20:4(n-6) substrate into cellular phosphatidylinositol and phosphatidic acid and a slower rise in the incorporation into phosphatidylcholine and phosphatidylethanolamine. Radioactivity was exclusively associated with the sn-2 position of each molecule. The labeling pattern of other phospholipids, neutral lipids, and eicosanoids was unchanged. TNF-alpha had no effect on the distribution of radioactivity in 1-acyl, 1-alkyl, and 1-alk-1-enyl subclasses of phosphatidylcholine, phosphatidylethanolamine, and triglyceride. Chain elongation, beta-oxidation and desaturation of [1-14C]20:4(n-6) were not modulated by the cytokine. TNF-alpha stimulated the release of [3H]20:4(n-6) from prelabeled neutrophils and also induced the production of endogenous unesterified 20:4(n-6). Concomitantly, treatment with the cytokine caused a decrease in the mass of cellular phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine and an increase in the levels of corresponding lysophospholipids, but had no significant effect on sphingomyelin, phosphatidic acid, diglyceride, and other lipids. TNF-alpha did not evoke neutrophils prelabeled with [3H]lyso platelet activating factor to produce [3H]phosphatidylethanol, [3H]phosphatidic acid, or [3H]diglyceride in the presence of ethanol, indicating that phospholipases D and C were not activated. Treatment of the leukocytes with the cytokine had no effect on the activity of neutral and acidic sphingomyelinase. These data collectively provide evidence that TNF-alpha specifically induces the turnover of neutrophil phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, which are enriched with 20:4(n-6) by the activation of phospholipase A2.