Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.