CD5 is a glycoprotein expressed on thymocytes, T cells, and a subset of B cells. Antibody-mediated cross-linking studies or studies on CD5 knockout mice implicate CD5 as a co-stimulatory or negative regulatory molecule. CD5 is rapidly phosphorylated on tyrosine (Y) residues following Tcell activation. Y429 and Y441 occur in an imperfect immunoreceptor tyrosine-based activation motif (ITAM)-like sequence. We investigated whether phosphatidylinositol (PI) 3-kinase, which binds to tyrosine-phosphorylated ITAM, interacts with CD5 following T cell activation. PI 3-kinase activity and the regulatory p85 subunit of PI 3-kinase associated with CD5 in pervanadate-stimulated, but not in unstimulated thymocytes. Cellular p85 as well as the recombinant Src homology 2 (SH2) domains of p85 bound a tyrosine-phosphorylated peptide encompassing Y463 with approximately threefold greater affinity than a doubly tyrosine-phosphorylated Y429-Y441 peptide. Binding of the C-SH2 domain to the Y463 phosphopeptide, together with preferential binding of the N-SH2 domain to the Y429-Y441 phosphopeptide, suggests a bivalent interaction. A 120-kDa phosphoprotein (pp120) associated with CD5 and specifically with the Y429-Y441 phosphopeptide in stimulated thymocytes. We conclude that stimulation of thymocytes with pervanadate induces the recruitment of PI 3-kinase and pp120 to CD5.