Activation of T lymphocytes often leads to cellular activation, production of cytokines, entry into cell cycle, and expression of Fas (CD95) and Fas ligand (FasL). Although it is well established that the interaction of Fas and FasL results in apoptosis, mechanisms for regulated expression of Fas and FasL are unclear. Our previous work with antisense oligodeoxynucleotides suggested that the protooncogene c-myc is obligatory for activation-induced apoptosis. To study the relationship between c-myc and the Fas/FasL expression, we employed the antisense method and a newly identified fungal metabolite, FR901228, which has been shown to specifically inhibit expression of c-myc in fibroblasts. We found that FR901228 could effectively block activation-induced apoptosis in T cell hybridomas and this was correlated with its specific inhibition of c-myc expression. Both FR901228 and antisense oligodeoxynucleotide to c-myc had similar effect in inhibiting FasL expression. These treatments did not affect activation-induced production of IL-2, nor the expression of Fas. In addition, FR901228 inhibited the expression of FasL in 3T3 fibroblasts, but not these transfected with c-myc, supporting a specific role of c-myc in this process. Thus, c-Myc plays a fundamental role in the regulation of the expression of FasL, but not Fas and IL-2. Our data further defined the requirement of c-Myc in activation-induced apoptosis in T cells.