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 perform was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40kDa 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. (C) 2002 Elsevier Science (USA). All rights reserved.