To explore the mechanism(s) by which the Syk protein tyrosine kinase participates in B cell antigen receptor (BCR) signaling, we have studied the function of various Syk mutants in B cells made Syk deficient by homologous recombination knockout. Both Syk SH2 domains were required for BCR-mediated Syk and phospholipase C (PLC)-gamma 2 phosphorylation, inositol 1,4,5-triphosphate release, and Ca2+ mobilization. A possible explanation for this requirement was provided by findings that recruitment of Syk to tyrosine-phosphorylated immunoglobulin (Ig) alpha and Ig beta requires both Syk SH2 domains. A Syk mutant in which the putative autophosphorylation site (Y518/Y519) of Syk was changed to phenylalanine was also defective in signal transduction; however, this mutation did not affect recruitment to the phosphorylated immunoreceptor family tyrosine-based activation motifs (ITAMs). These findings not only confirm that both SH2 domains are necessary for Syk binding to tyrosine-phosphorylated Ig alpha and Ig beta but indicate that this binding is necessary for Syk (Y518/519) phosphorylation after BCR ligation. This sequence of events is apparently required for coupling the BCR to most cellular protein tyrosine phosphorylation, to the phosphorylation and activation of PLC-gamma 2, and to Ca2+ mobilization.