Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor γ-chain (FcR γ-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-γ2 (PLC-γ2). In this study tyrosine-phosphorylated proteins that associate with PLC-γ2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-γ2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-γ2. The majority of the tyrosine-phosphorylated proteins that associate with PLC-γ2 bind to its C-terminal SH2 domain. These were found to include PLC-γ2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR γ-chain. Direct association was detected between PLC-γ2 and SLP-76, and between PLC-γ2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR γ-chain and Lyn were found to co-immunoprecipitate with PLC-γ2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-γ2 in platelets is in contrast with that for PLC-γ1 and Syk in B cells. The in vivo function of PLC-γ2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-γ2. These data indicate that the C-terminal SH2 domain of PLC-γ2 is important for PLC-γ2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR γ-chain.