The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.