T helper (Th) 2-type cytokines play a dominant role in allergic inflammation. Accumulating evidence suggests that Th1-type cytokines antagonize Th2-type cytokine responses; however, recent studies demonstrate that Th1 cytokines might enhance Th2 immune responses. We examined whether interferon (IFN)-gamma, a representative Th1 cytokine, modifies the effector functions of human eosinophils stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5. GM-CSF and IL-5 have significant functional homology, and contribute to the regulation of Th2 immunity. After the pretreatment of eosinophils with IFN-gamma, GM-CSF- or IL-5-induced eosinophil functions were examined, including superoxide anion generation, degranulation, adhesion, expression of GM-CSF receptor (R), IL-5R, or CD11b, and phosphorylation of intracellular signaling molecules. Superoxide anion generation was measured using the cytochrome c reduction method. Degranulation and cell adhesion were evaluated based on eosinophil-derived neurotoxin (EDN) contents in supernatants or adherent cells. Phosphorylation of signaling molecules was analyzed using a multiplex beads array system. Preincubation with IFN-gamma resulted in enhanced GM-CSF- or IL-5-induced superoxide anion generation and degranulation of human eosinophils, whereas stimulus-induced eosinophil adhesion was unaffected. In addition, IFN-gamma did not influence the expression of GM-CSFR, IL-5R, and CD11b. Furthermore, IFN-gamma upregulated GM-CSF- or IL-5-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and activating transcription factor (ATF)-2. Finally, we confirmed that MAPK inhibitors blocked the enhancement of stimuli-induced superoxide anion generation of IFN-gamma treated eosinophils. In conclusion, IFN-gamma might upregulate ERK, p38, or JNK/ATF-2 phosphorylation induced by GM-CSF or IL-5, leading to enhanced cytokine-induced eosinophil superoxide generation and degranulation.