Cytotoxic T lymphocytes (CTL) specific for human minor histocompatibility (H) antigens can be isolated from the blood of major histocompatibility complex (MHC)-matched allogeneic bone marrow transplant (BMT) recipients and may play a prominent role in the graft-versus-host (GVH) and graft-versus-leukemia (GVL) reactions (Tsoi et al, J Immunol 125:2258, 1980; Tsoi et al, Transplant Proc 15:1484, 1983; Goulmy et al, Nature 302:159, 1983; Irle et al, Transplantation 40:329, 1985; and Niederwieser et al, Blood 81:2200, 1993). The identification of minor H antigens that are expressed in hematopoietic cells, including leukemic cells, but not in fibroblasts and other tissue types has suggested that such tissue-restricted antigens could potentially serve as targets for T-cell immunotherapy to enhance GVL activity without inducing GVH disease (de Bueger et al, J Immunol 149:1788, 1992; van der Harst et al, Blood 83:1060, 1994; and Dolstra et al, J Immunol 158:560, 1997). To explore the feasibility of this strategy, donor CD3+CD8+ CTL clones specific for recipient minor H antigens were isolated and characterized from allogeneic BMT recipients. CTL clones were obtained from the majority of donor/recipient pairs. Seventeen distinct minor H antigens distinguishable by their MHC-restricting allele, population frequency, and/or distribution of tissue expression were defined by 56 CD3+CD8+ CTL clones isolated from these patients. The MHC-restricting alleles for these CTL clones included HLA-A2 and HLA-B7, which had previously been shown to present minor H antigens to CTL, as well as HLA-A3, -A11, -B8, -B53, and -Cw7, which had not previously been described to present minor H antigens to CTL. Estimated phenotype frequencies for these 17 distinct minor H antigens range from 0.17 to 0.92. In vitro cytotoxicity assays using hematopoietic cells and fibroblasts as target cells showed that 5 of the 17 minor H antigens were expressed in both hematopoietic cells and fibroblasts. However, 12 were presented for CTL recognition only by hematopoietic cells and not by dermal fibroblasts derived from the same donors. These results significantly extend the spectrum of CTL-defined human minor H antigens that could potentially serve as target antigens for cellular immunotherapy to promote GVL activity after allogeneic BMT.