In T lymphocytes, several proteins are rapidly phosphorylated on tyrosine after stimulation. In this study we examine the ability of tyrosine phosphorylated proteins from Jurkat T cells stimulated by CD2 or T cell receptor-CD3 to interact with the src homology 2 or src homology 3 domains from eight different proteins involved in signal transduction in lymphocytes: Vav, Shc, Nck, phosphatidylinositol-3-kinase, phospholipase C-gamma 1, Ras-GTPase activating protein, c-Crk and Grb2. Our data show that different SH2 domains have distinct patterns of binding to phosphotyrosine containing proteins. We show that Vav, a protein expressed only in hematopoietic cells that may have guanine nucleotide releasing factor activity, is able to interact with certain SH2-containing proteins depending on its tyrosine phosphorylation and with Grb2 in a manner independent of phosphorylation on tyrosine. Coimmunoprecipitation experiments support the idea of a trimolecular complex Shc-Grb2-Vav in vivo. These data suggest a central role played by Vav and provide insight in the complexity and specificity of protein-protein interactions in the signaling events in lymphocytes.