Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.