Engagement of peptide-MHC by the TCR induces a conformational change in CD3epsilon that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3epsilon ITAM, has been implicated in synapse formation and T cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3epsilon PRS independent. In this study, we show that the CD3epsilon PRS is necessary for peptide-MHC-induced phosphorylation of CD3epsilon and for recruitment of protein kinase Ctheta to the immune synapse in differentiated CD8+ T lymphocytes. However, whereas these two events are dispensable for functional T cell responsiveness to high-avidity ligands, they are required for responsiveness to low-avidity ones. Thus, in at least certain T cell clonotypes, the CD3epsilon PRS amplifies weak TCR signals by promoting synapse formation and CD3epsilon phosphorylation.