Rapamycin (RAPA), an inhibitor of cytokine responses, is under investigation in humans for graft-vs-host disease (GVHD) prevention. The mechanisms responsible for GVHD prevention are unknown. We show that RAPA is more effective in inhibiting CD8+ or TCR γδ+ than CD4+ T cell-mediated murine GVHD. To determine how RAPA inhibited GVHD, thoracic duct lymphocytes (TDL) were isolated from recipients of allogeneic donor grafts. Compared with controls, RAPA-treated recipients had a marked decrease in donor TDL T cell number between days 5 and 24 posttransplant. CD8+ T cell expansion was preferentially inhibited. RAPA inhibited Th1 or Th1 cytotoxic (Tc1) cytokines, but not Th2 or Tc2, cell generation. In situ mRNA hybridization also showed that TDL T cells from RAPA-treated mice had a lower frequency of granzyme B+ cells, indicating that RAPA inhibited the generation of CTL capable of mediating cytolysis through the release of granzyme B. In another system, RAPA was found to inhibit the GVL response of delayed donor lymphocyte infusions. Since CD8+ T cells are the primary effectors in this system, these data suggest that RAPA directly interfered with GVL effector cell expansion or function. We conclude that RAPA is effective in inhibiting Th1 or Tc1 cytokine production and CD8+ and TCRγδ+ T cell-mediated GVHD, but abrogates GVL.