Lymphocyte-mediated cytotoxicity represents one defense mechanism that contributes to transplant rejection. Comparatively little is known about the molecular mechanisms responsible for cell-mediated rejection of xenografts. Herein, we have investigated the relative contribution of perforin- and Fas- pathways in lymphocyte-mediated cytotoxicity generated in response to a variety of human cell lines and peripheral blood mononuclear cells transplanted into mice. Responder lymphocytes generated in immunocompetent mice displayed significant lysis of human targets, suggesting that normally mice can generate a strong lymphocytotoxic response to human cells. Effector cells from mice immunized with one human cell line were also cytotoxic to other human cells, indicating a cross-reactive mouse antihuman response. Effector cells generated in gld mice that have a mutated Fas ligand displayed apparently normal levels of cytotoxicity against human target cells, suggesting a predominantly perforin-based cytotoxic mechanism. This was confirmed by the low cytotoxic activity of xenospecific lymphocytes from perforin-deficient mice. The residual cytotoxicity in perforin-deficient mice responding to xenografted human cells was completely inhibited by anti-Fas mAb, suggesting that a Fas-mediated pathway can be stimulated in the absence of perforin. The detection of Fas-mediated cytotoxicity correlated with the sensitivity of human target cells to Fas-mediated lysis. Depletion of effector CD8+ T, but not CD4+ T or NK1.1+, cells almost completely inhibited lysis of human target cells, suggesting that CD8+ T cells were responsible for perforin-mediated xenospecific cytotoxicity. Overall, these data suggested that xenospecific cytotoxic T lymphocytes can lyse target cells via either perforin- or Fas-mediated pathways.