T lymphocytes require a signal via their antigen specific receptors (the T cell receptors) and an antigen independent costimulatory signal. Signals through CD28 can costimulate T cells in the presence of limiting amounts of T cell receptor signal, or in the presence of PMA, providing this second signal. CD28 signaling is known to involve the activation of protein tyrosine kinases. Using the Jurkat leukemic cell line as a model, we have tested CD28 crosslinking for its effects on the protein tyrosine kinases p56lck. We report that following crosslinking of CD28, p56lck kinase activity is increased. Crosslinking CD28 causes a shift in the relative mobility of p56lck from 56 to 60 kD similar to that seen after crosslinking of CD2 and CD4, cell surface receptors known to be associate with and activate p56lck. Finally, lck could be found in anti-CD28 immunoprecipitates in exponentially growing Jurkat and in "activated" CD28 (i.e., cross linked), but not in CD28 in resting Jurkat cells. These findings suggest an important role for p56lck in CD28 signal transduction.