The present study was undertaken to define the cellular mechanisms involved in the rejection of major histocompatibility complex (MHC) class I disparate skin grafts by mice depleted of CD8+ T cells in vivo. Mice were effectively depleted of CD8+ T cells by adult thymectomy followed by in vivo administration of anti-CD8 monoclonal antibody (mAb) and then engrafted with allogeneic skin. We found that CD8 depleted mice did reject MHC class I disparate skin grafts, but only when the grafts also expressed additional alloantigens. Despite the marked depletion of CD8+ T cells in these mice, we found that their rejection of MHC class I disparate grafts was mediated by CD8+ cytolytic T lymphocyte (CTL) effectors that had escaped depletion. These CD8+ CTL effectors were unique in that: (a) their generation was dependent upon the injected anti-CD8 mAb and upon exposure to class I MHC alloantigens expressed on the engrafted skin, and (b) their effector function was resistant to blockade by anti-CD8 mAb. We observed that the additional alloantigens coexpressed on MHC class I disparate grafts that triggered graft rejection in CD8-depleted mice could be MHC-linked or not and that they functioned in these rejection responses to activate third party specific CD4+ T helper (Th) cells to provide helper signals for the generation of CD8+ anti-CD8 resistant CTL effector cells. Thus, mice depleted of CD8+ T cells by thymectomy and in vivo administration of anti-CD8 mAb harbor a unique population of anti-CD8 resistant, CD8+ effector cells that mediate anti-MHC class I responses in vivo and in vitro, but require help from third party specific Th cells to do so.