Stimulation of the T cell antigen receptor (TCR).CD3 complex induces rapid tyrosine phosphorylation of Cbl, a protooncogene product which has been implicated in intracellular signaling pathways via its interaction with several signaling molecules. We found recently that Cbl associates directly with a member of the 14-3-3 protein family (14-3-3tau) in T cells and that the association is increased as a consequence of anti-CD3-mediated T cell activation. We report here that phorbol 12-myristate 13-acetate stimulation of T cells also enhanced the interaction between Cbl and two 14-3-3 isoforms (tau and zeta). Tyrosine phosphorylation of Cbl was not sufficient or required for this increased interaction. Thus, cotransfection of COS cells with Cbl plus Lck and/or Syk family protein-tyrosine kinases caused a marked increase in the phosphotyrosine content of Cbl without a concomitant enhancement of its association with 14-3-3. Phorbol 12-myristate 13-acetate stimulation induced serine phosphorylation of Cbl, and dephosphorylation of immunoprecipitated Cbl by a Ser/Thr phosphatase disrupted its interaction with 14-3-3. By using successive carboxyl-terminal deletion mutants of Cbl, the 14-3-3-binding domain was mapped to a serine-rich 30-amino acid region (residues 615-644) of Cbl. Mutation of serine residues in this region further defined a binding motif distinct from the consensus sequence RSXSXP, which was recently identified as a 14-3-3-binding motif. These results suggest that TCR stimulation induces both tyrosine and serine phosphorylation of Cbl. These phosphorylation events allow Cbl to recruit distinct signaling elements that participate in TCR-mediated signal transduction pathways.