CD28, a T cell costimulatory receptor, provides a signal that induces both optimal proliferation and the production of IL-2 by TCR-activated T cells. We show that the stimulation of CD28 leads to the activation of p21-activated kinase and MEK kinase 1. The same pathway was also stimulated in T cells treated with the cell-permeable ceramide analogue, C2-ceramide. The combined stimulation of either CD3 and CD28 or CD3 concurrently with C2-ceramide largely enhanced the activity of p21-activated kinase and MEK kinase 1. Therefore the Rac1/CDC42-coupled pathway (s) is a candidate that transduces and facilitates cross-talk between the CD28 costimulatory signal and the TCR signal.