Cytotoxic T-cells (CTLs) specific for the hematopoietic system-restricted minor histocompatibility antigen (mHag) HA-1 efficiently lyse HA-1-positive leukemic cells without affecting nonhematopoietic cells. HA-1-specific CTLs are thus potential tools for adoptive immunotherapy of relapsed leukemia after HLA-matched-HA-1-mismatched stem cell transplantation (SCT). In vitro generation of HA-1-specific CTLs from SC donors is possible using dendritic cells (DCs) pulsed with synthetic HA-1 peptide as stimulator cells. However, this approach requires at least 6 weeks of in vitro culturing under GMP (good manufacturing practice) conditions. Our data show that in vitro induction of HA-1-specific CTLs is more rapid with the use of DCs that are retrovirally transduced with the HA-1 complementary DNA. Retrovirally transduced DCs showed functional and long-term stable expression of the HA-1 CTL epitope in primary CTL cultures. In 4 SC donors, HA-1-transduced DCs induced HA-1-specific CTLs in 14 to 21 days. The in vitro-generated CTL lines contained 6% to 9% T-cells that stained brightly with tetrameric HLA-A2/HA-1 peptide complexes (HA-1(A2) tetramer) and showed significant lysis of HA-1+ leukemic cells. The CTL induction procedure using peptide-pulsed DCs was less effective and required 28 to 35 days of T-cell culture. Thus, sustained presentation of mHag HA-1 by retrovirally transduced DCs facilitates the in vitro induction of HA-1-specific CTLs.