Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B 4 and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca 2؉ ] i induced by thimerosal in neutrophils stimulated with GM-CSF/ fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca 2؉ ] i was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A 2 ␣ activation.Neutrophils play an important role in natural immunity and function to eliminate microbes and dead tissues. They are also a major cell involved in the acute inflammatory response, since they respond rapidly to chemotactic stimuli and can be activated by cytokines produced primarily by macrophages to perform degradative and phagocytic functions. Upon activation at the site of inflammation, they release lysosomal enzymes and neutral proteinases, reactive oxygen species, including H 2 O 2 and O 2 .(1, 2), and metabolites of arachidonic acid (AA), 2 in particular leukotriene B 4 (LTB 4 ) (3). Biosynthesis of leukotrienes involves the release of AA from membrane phospholipids by Ca 2ϩ -dependent cytosolic phospholipase A 2 ␣ (cPLA 2 ␣) (4) and its conversion into 5-hydroperoxyeicosatetraenoic acid and subsequently leukotriene A 4 (LTA 4 ), catalyzed by 5-lipoxygenase (5-LO) (5). LTA 4 is then enzymatically converted to LTB 4 by LTA 4 -hydrolase (6) or to leukotriene C 4 (LTC 4 ) by the addition of a molecule of glutathione through the action of LTC 4 -synthase (7). The site for most of these biochemical events is the perinuclear region (8).Regulation of leukotrie...