CD28 is a costimulatory receptor that can provide the second signal necessary for T-cell activation and function in response to stimulation through the T-cell antigen receptor/CD3 complex. We found that a distinct array of proteins was phosphorylated on tyrosine following stimulation with anti-CD28 monoclonal antibody, as detected by immune-complex kinase assays. Anti-CD28 stimulation of in vitro kinase activity was detergent-dependent, occurring in immune complexes prepared with Brij 96 but not Nonidet P-40. Pretreatment of cells with low concentrations of phorbol ester increased the activation-independent phosphorylation of proteins in CD28 immune complexes. Reimmunoprecipitation studies indicated that the cytoplasmic protein-tyrosine kinases Lck and Fyn were associated with CD28. CD28 itself was phosphorylated both in vitro and in vivo in an activation-dependent manner, as detected by nonreducing/reducing SDS/PAGE analyses. The activation-stimulated phosphorylation of CD28 may play a key role in signaling through this receptor.