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Annexin Al (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrowderived mast cells. This cleavage event was inhibited when intracellular Ca2+ signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A2 (cPLA2) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA2 interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA2 mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA2 and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA2 and production of eicosanoids during mast-cell activation. [ABSTRACT FROM AUTHOR]