As little is known about the molecular mechanisms responsible for lymphocyte-mediated rejection of xenografts, we have studied the relative contribution of perforin and Fas pathways in cytotoxic lymphocytes generated in mice transplanted with human cell lines. Responder lymphocytes generated in immunocompetent mice displayed significant lysis of human target cells, which suggests that mice can generate a strong lymphocytotoxic response to human cells. Effector cells generated in immunocompetent andgld(Fas ligand mutant) mice predominantly use a perforin-mediated cytotoxic mechanism. By contrast, a Fas-mediated pathway could be stimulated in perforin-deficient or β2-microglobulin-deficient mice, providing the human target cells were sensitive to Fas-mediated lysis. In vitro depletion of effector CD3+CD8+T cells, but not CD4+T or NK1.1+cells, completely inhibited lysis of human target cells. This suggests that CD3+CD8+T cells were responsible for perforin-mediated xenospecific cytotoxicity. In vivo depletion of NK1.1+cells and CD4+T cells before the final immunization abrogated the capacity of lymph node cells to generate xenospecific CD8+cytotoxic T lymphocytes. By contrast, in vitro depletion of CD4+T cells was most effective in abrogating the xenospecific Fas-mediated cytotoxicity of perforin-deficient effector cells. Xenospecific cytotoxic T cells were also capable of mediating tumor rejection when adoptively transferred intoscid/scidmice bearing established human COLO 205 xenografts. Overall, these data suggested that xenospecific cytotoxic T lymphocytes can lyse target cells via either perforin- or Fas-mediated pathways and that these cells can provide protective and specific immunity against tumor xenografts in the absence of an intact humoral immune system.