Mast cells and T cells produce reactive oxygen species (ROS) after stimulation with the high-affinity IgE receptor (Fc epsilon RI) and T cell receptor. A growing body of evidence suggests the existence of ROS-regulated intracellular and/or plasma membrane Ca(2+) channels in these cells but their molecular entities remain to be identified. Here, we report that store-operated Ca(2+) channel (SOC) activity is regulated by superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)) in mast cells. MnTBaP (Mn(III)tetrakis(4-benzoic acid)porphyrin) and ebselen (2-phenyl-1,2-benziso-selenazol-3(2H)-one) selectively blocked the generation of O(2)(*-) and H(2)O(2), respectively, in antigen-stimulated cells. The H(2)O(2) generation was dependent on the Src family kinase (SFK) and phosphatidylinositol-3-kinase (PI3K) activities but independent of extracellular Ca(2+), and the Fc epsilon RI beta-chain immunoreceptor tyrosine-based activation motif played an essential role. On the other hand, O(2)(*-) generation was strictly dependent on extracellular Ca(2+), but negatively regulated by the SFK and PI3K activities. Inhibition of O(2)(*-) generation resulted in increased H(2)O(2) generation and reduced SOC activity, although it had a minimal effect on endoplasmic reticulum Ca(2+) store depletion. On the contrary, inhibition of H(2)O(2) generation resulted in increased intracellular O(2)(*-) generation and augmented SOC activity. The findings suggest that O(2)(*-) and H(2)O(2), which are generated by separate signaling pathways/sources, reciprocally regulate SOC activity in mast cells. Such generations of multiple oxidant species and their distinct roles in the regulation of SOC activity may facilitate the fine tuning of Ca(2+) signaling in mast cells.