Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg⁵²⁵ to Gln), Src homology 2 (SH2, Arg³⁰⁷ to Ala), or pleckstrin homology (PH, Arg²⁸ to Cys) domains, we investigated the roles and structure−function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP₃ production and the significantly reduced tyrosine phosphorylation of PLCγ2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP₃ generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCγ2 and catalyze IP₃ production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCγ2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCγ2 through BLNK in order to position the Btk, PLCγ2, and phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLCγ2 and to maximize its catalytic efficiency for IP₃ production.