Recent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2(-) donors by sequential stimulation with peptide-pulsed HLA-A2(+) stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2(+) patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2(+) patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2(+) fibroblasts or HLA-A2(-) normal PBMCs. Coculture of CD34(+) PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit-granulocyte macrophage (CFU-GM) formation in HLA-A2(+) healthy controls and CML patients but resulted in no significant inhibition in HLA-A2(-) healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that adoptive immunotherapy with allorestricted CTLs directed against CD45 epitopes may be useful in restoring the GVL effect after HLA-A2-mismatched haploidentical transplantation. Further, because P1218-specific CTLs also recognize healthy HLA-A2(+) progenitors, such CTLs could also contribute to host myeloablation and enhance donor cell engraftment.