Antigenic cross-linking of the high affinity IgE receptor (FcεR1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation of the SH2 and SH3 domain containing adapter molecule Grb2 by FcεR1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S-transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL,2H3. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for FcεR1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2 may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a 33- kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and FcεR1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc(R1-activated PTKs and binds to Grb2 SH2 domains in FcεR1 activated but not quiescent cells. The β subunit of the FcεR1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2- binding protein described in the present report is not the FcεR1 β chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein complexes in mast cells of which a subset are FcεR1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to FcεR1 ligation: the 75- kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. We propose that protein complex formation by Grb2 is an important consequence of FcεR1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to bring about optimal mast cell end function.