We have established a pre-B acute lymphoblastic leukemia (ALL) cell line, NAGL-1, from the bone marrow of a patient diagnosed with pre-B ALL. The patient has been disease-free for the 4 years since allogeneic bone marrow transplantation from her HLA-genotypically identical sister. NAGL-1 showed a pre-B cell phenotype (CD19+, CD10+, c mu+, s mu-) mostly identical to freshly isolated leukemic cells from the patient. This cell line strongly expressed HLA class I and HLA-DR molecules, as well as the costimulatory molecules CD54, CD40, and CD86. Cytotoxic T-lymphocyte (CTL) lines were generated by stimulating the donor-derived peripheral blood mononuclear cells with either irradiated leukemic cells or NAGL-1. Both CTL lines showed specific lysis against NAGL-1 in 51Cr release assays. Lytic activity was partially inhibited by anti-CD8 and anti-HLA class I monoclonal antibodies. Treatment of NAGL-1 with TNF-alpha increased its susceptibility to the CTL line. One CD8+ T cell clone derived from the CTL line killed both the patient phytohemagglutinin (PHA) blasts and NAGL-1 but not the donor PHA blasts, suggesting that the clone recognized the patient-specific minor antigen presented on both PHA blasts and NAGL-1. Utilization of leukemic cell lines could be a useful model for the development of CTL lines and clones for immunological study and potential immunotherapy.