Within a few minutes of T-cell activation, transcription of a set of genes including c-fos and c-jun is activated. For maximal induction of c-jun, at least two major signal pathways are required. One can be triggered by T-cell receptor engagement or phorbol esters and the other by anti-CD28 engagement. The c-jun promoter region between -117 and -50 contains binding sites for the transcription factors Spl, CTF, ATF/CREB, and MEF2. In this study, we sought to map the sequences in the c-jun promoter responsible for CD28-mediated induction in activated Jurkat T cell by point mutational analysis. We found that mutation of the c-jun MEF2 site strongly reduces CD28 induction of the promoter in Jurkat T cells and that MEF2D is the major binding molecule to the c-jun MEF2 site in Jurkat T cells. Mutation of the c-jun ATF site also partially reduced CD28 induction of the promoter. In addition, pretreatment with an endolysomotropic agent NH4Cl, an acidic sphingomyelinase inhibitor, completely inhibited the activation of the c-jun promoter by anti-CD28 antibody treatment, whereas pretreatment with wortmannin, a PI3-kinase inhibitor, did not affect the induction of the c-jun promoter. These results suggest that CD28 signaling leading to the c-jun promoter involves acidic sphingomyelinase, but not PI3-kinase, to activate factors binding to the MEF2 and ATF sites.