Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which is assumed to be achieved through the activation of phospholipase Cγ2 (PLCγ2) via a tyrosine phosphorylation mechanism in B cells. Here we show that H₂O₂ induces both tyrosine phosphorylation on PLCγ2 and the activation of phosphatidylinositol 3-kinase (PI3K) in B cells, and that the phosphatidylinositol 3-kinase inhibitor, Wortmannin, partially inhibited the H₂O₂-induced calcium release without affecting tyrosine phosphorylation on PLCγ2. Overexpression of human Bruton's tyrosine kinase (Btk), which was activated by H₂O₂, almost completely overcame the inhibition of calcium release by Wortmannin. The reversal of Wortmannin's inhibition by enhancing Btk concentration seemed unique to the H₂O₂-mediated effect, because Btk failed to overcome the inhibition of Wortmannin on B cell receptor-triggered calcium mobilization. Immunoblot analysis revealed that Btk formed stable complexes with several tyrosine-phosphorylated proteins, including PLCγ2, only in Btk-overexpressed cells on H₂O₂ stimulation. Together, our data are consistent with the notion that PIP3 and/or a high concentration of Btk target the activated PLCγ2 to its substrate site for maximal catalytic efficiency.