At present, allogeneic bone marrow transplantation (BMT) is the only curative treatment for chronic myeloid leukemia (CML) in chronic phase (CP). The graft-vs.-leukemia (GVL) effect appears to play an important role in this treatment. Direct evidence for a GVL effect has been reported in Ph1-positive CML patients who relapsed after allogeneic BMT and who were treated with leukocyte transfusion from the original marrow donor. Alpha-interferon (alpha-IFN) may have facilitated this GVL effect since many patients were treated with it also. We investigated whether leukemia-reactive cytotoxic T lymphocytes (CTLs) can be generated from human leukocyte antigen (HLA)-genotypically identical sibling bone marrow (BM) donors who donated marrow for two patients with Ph1-positive CML in CP and one patient with Ph1-positive acute lymphoblastic leukemia (ALL). We also investigated alpha-IFN's ability to facilitate the generation of CTLs. In the absence of alpha-IFN, CTL lines with only low cytotoxicity and no CTL clones could be generated. In the presence of alpha-IFN, however, alloreactive, leukemia-reactive CTL lines with high cytotoxicity could be generated, and CD8+ CTL clones could be established with HLA class I restricted minor histocompatibility antigen (mHa)-specific recognition. In a cell-mediated clonogenic cytotoxicity assay, the CTL clones showed specific growth inhibition of leukemic precursor cells from the recipient and a second CML patient, but the clones did not inhibit growth of hematopoietic precursor cells (HPCs) from the donor. The normal HPCs from an unrelated donor with the HLA class I restriction molecule were also recognized by the CTL clones, illustrating that the antigen recognized is not leukemia-specific. The mechanism of the immunomodulating effect by alpha-IFN is not clear. Addition of alpha-IFN to medium did not alter the expression of HLA or adhesion molecules on CML cells. In the treatment of CML, administration of alpha-IFN as adjuvant immunotherapy after allogeneic BMT may increase GVL reactivity.