T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3'-kinase (PI 3-kinase). In a previous report, we showed that the p85alpha subunit of the PI 3-kinase preferentially associated with the CD3-zeta membrane-proximal immunoreceptor tyrosine-based activation motif of the zeta chain (zetaA-ITAM) (Exley, M., Varticovski, L., Peter, M., Sancho, J., and Terhorst, C. (1994) J. Biol. Chem. 269, 15140-15146). Here, we demonstrate that tyrosine phosphorylation of CD3-epsilon can recruit the PI 3-kinase enzyme in a T cell activation-dependent manner. In vivo studies with Jurkat cells stably transfected with a CD8-CD3-epsilon chimera (termed CD8-epsilon) shows that ligation of endogenous CD3-epsilon or CD8-epsilon by specific antibodies induces tyrosine phosphorylation of CD3-epsilon or CD8-epsilon, respectively. Increased tyrosine phosphorylation correlates with increased binding of p85alpha PI 3-kinase and recruitment of PI 3-kinase enzymatic activity to CD3-epsilon or CD8-epsilon proteins. Mutagenesis studies in COS-7 cells, transiently transfected with CD8-epsilon, p85alpha, and Fyn cDNAs in various combinations, show that both Tyr170 and Tyr181 within the CD3-epsilon-ITAM are required for efficient binding of p85alpha PI 3-kinase. Thus, replacement of Tyr170 by Phe (Y170F), or Tyr181 by Phe (Y181F) significantly reduces binding of p85alpha PI 3-kinase, whereas it does not affect binding of Fyn. Further in vitro experiments suggest that a direct binding of the tandem SH2 domains of p85alpha PI 3-kinase to the two phosphorylated tyrosines in a single CD3-epsilon-ITAM may occur. The data also support a model in which a single CD3 subunit can recruit distinct effector molecules by means of TCR-mediated differential ITAM phosphorylation.