p95vav, the product of the vav protooncogene, has been implicated in the T cell receptor (TCR)-mediated signaling cascade p95vav is phosphorylated on tyrosine residues after TCR stimulation by anti-TCR/CD3 antibodies and possesses a number of landmark features of signaling molecules such as a putative guanine nucleotide exchange factor domain, a pleckstrin homology domain, and an Sre homology (SH) 2 and two SH3 domains, which provide the capacity to form multimeric signaling complexes. However, the precise role of p95vav in TCR signaling remains unclear. In this work we show that physiological stimulation of T cell hybridomas with antigen presented by major histocompatibility complex class II molecules leads to a strong tyrosine phosphorylation of p95vav and its association with tyrosine-phosphorylated SLP-76. SLP-76 is a newly described SH2-containing protein that has been previously found to bind to the adapter molecule Grb2. Moreover, we provide evidence that p95vav-SI P-76 association is SH2-mediated by demonstrating that this interaction can be inhibited by a phosphopeptide containing a putative p95vav-SH2-binding motif (pYESP) present in SLP-76. Furthermore, in vitro experiments show that after antigen stimulation, phosphorylated p95vav-SLP-76 can bind to Grb2 in a complex that contains pp36/38 and pp116 proteins. Our data provide a clue to explain recent independent observations that overexpression of p95vav or SLP-76 enhances TCR-mediated gene activation.