Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non-receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine-phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation-dependent phosphoproteins in vitro. SH2 domains from Src-like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP-70 tyrosine kinase bound to phosphorylated Ig-β but only weakly to Ig-α. Also the SHC-derived SH2 domain formed complexes with the tyrosine-phosphorylated Ig-α/β heterodimer, while the C- and N-terminal SH2 domains of GTPase-activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2-phosphotyrosine-mediated manner. The data also provide a possible explanation for the notion that Ig-α and Ig-β might couple to different biochemical pathways.