Donor lymphocyte infusion (DLI) may lead to lymphohematopoietic graft-versus-host (GVH) reactions without graft-versus-host-disease (GVHD). The graft-versus-leukemia (GVL) effect of DLI is greater in mixed chimeras than in full chimeras following fully MHC-mismatched bone marrow transplantation (BMT). Some studies showed, following MHC-mismatched BMT, that only CD8+ T cells are involved in GVL effects of DLI against MHC class I+ class II− tumors, but others showed that the GVL effect of DLI is dependent on both CD8+ and CD4+ T cells. Previously, we have observed that GVL against EL4 is mediated by CD8+ T cells but not CD4+ T cells following DLI in freshly irradiated mice treated with cytokines to reduce GVHD mortality in fully MHC-mismatched BMT. The exact contribution of CD4+ and CD8+ T cells to the GVL effects of DLI in established mixed chimeras is not known. We hypothesized that lethal conditioning leads to the production of many proinflammatory cytokines which may help to generate a CD4 cell-independent, CD8 cell-mediated anti-tumor effect. However, in delayed DLI recipients, CD4+ helper T cells might be needed to induce CD8+ anti-tumor effector cells. We evaluated the role of T-cell subsets in GVL effects of DLI following fully major histocompatibility complex (MHC)-mismatched BMT in freshly irradiated mice and mixed chimeric delayed DLI recipients. While GVL effects of delayed DLI given to established mixed chimeras were dependent on both CD4+ and CD8+ T cells, CD4+ T cells did not contribute to GVL effects in freshly irradiated mice. Consistent with this, MHC class II expression on host antigen-presenting cells (APCs) was needed to optimize GVL effects of DLI in established mixed chimeras. To address whether cross-presentation of tumor antigens (Ag) via class I would permit GVL responses in full chimeras, we generated full chimeras in which donor and recipient shared a class I locus. However, there were no GVL effects of delayed DLI in full chimeras, providing no evidence that cross-presentation of tumor Ag through class I molecules shared by donor and the tumor led to anti-tumor effects in this model. We conclude that following delayed DLI to established mixed chimeras, the GVL response requires extensive MHC disparity, MHC class II expression on host APCs and the presence of CD4+ T cells. In contrast, the requirement for CD4+ T cells can be bypassed in freshly irradiated recipients, but this occurs at the expense of a greater risk of GVHD. Administration of delayed DLI to mixed chimeras permits achievement of optimal GVL effects because large numbers of GVH-reactive T cells can be administered without inducing GVHD.