Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which is assumed to be achieved through the activation of phospholipase C gamma 2 (PLC gamma 2) via a tyrosine phosphorylation mechanism in B cells. Here we show that H2O2 induces both tyrosine phosphorylation on PLC gamma 2 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 PLC gamma 2. 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 PLC gamma 2, 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 PLC gamma 2 to its substrate site for maximal catalytic efficiency.