In addition to the well-recognized ability of prostaglandin D2 (PGD2) to regulate eosinophil trafficking, we asked whether PGD2 was also able to activate eosinophils and control their leukotriene C4 (LTC4)-synthesizing machinery. PGD2 administration to presensitized mice enhanced in vivo LTC4 production and formation of eosinophil lipid bodies-potential LTC4-synthesizing organelles. Immunolocalization of newly formed LTC4 demonstrated that eosinophil lipid bodies were the sites of LTC4 synthesis during PGD2-induced eosinophilic inflammation. Pretreatment with HQL-79, an inhibitor of PGD synthase, abolished LTC4 synthesis and eosinophil lipid body formation triggered by allergic challenge. Although PGD2 was able to directly activate eosinophils in vitro, in vivo PGD2-induced lipid body-driven LTC4 synthesis within eosinophils was dependent on the synergistic activity of endogenous eotaxin acting via CCR3. Our findings, that PGD2 activated eosinophils and enhanced LTC4 synthesis in vivo in addition to the established PGD2 roles in eosinophil recruitment, heighten the interest in PGD2 as a target for antiallergic therapies.