Eosinophils and basophils, when activated, become major sources of cysteinyl leukotrienes, eicosanoid mediators pertinent to allergic inflammation. We show that the C-C chemokines, eotaxin and RANTES (regulated upon activation normal T cell expressed and secreted), activate eosinophils and basophils for enhanced leukotriene C(4) (LTC(4)) generation by distinct signaling and compartmentalization mechanisms involving the induced formation of new cytoplasmic lipid body organelles. Chemokine-induced lipid body formation and enhanced LTC(4) release were both mediated by CCR3 receptor G protein-linked downstream signaling involving activation of phosphoinositide 3-kinase, extracellular signal-regulated kinases 1 and 2, and p38 mitogen-activated protein kinases. Chemokine-elicited lipid body numbers correlated with increased calcium ionophore-stimulated LTC(4) production; and as demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid, lipid bodies were the predominant sites of LTC(4) synthesis in both chemokine-stimulated eosinophils and chemokine-primed and ionophore-activated eosinophils. Eotaxin and RANTES initiated signaling via phosphoinositide 3-kinase and mitogen-activated protein kinases both elicits the formation of lipid body domains and promotes LTC(4) formation at these specific extranuclear sites.