Active specific immunotherapy targeting tumor-associated antigens (TAA) requires reagents of high immunogenicity and safety. To address this issue, we constructed a recombinant vaccinia virus carrying a minigene insert encoding the HLA-A2.1-restricted MART-1/Melan-A27-35 melanoma TAA (rVV-M). To facilitate the entry of the antigenic epitope into the endoplasmic reticulum, a sequence coding for adenovirus E3/19K leader peptide was added. This rVV-M was made replication-incompetent by treatment with psoralen and UV light. Infection with rVV-M rendered HLA-A2.1 EBV-transformed lymphoblastoid cells sensitive to the cytotoxic effects of HLA-class-1-restricted, MART-1/Melan-A27-35-specific cytotoxic T lymphocytes (CTL). The capacity of rVV-M to generate HLA-A2.1-restricted MART-1/Melan A-specific CTL was demonstrated from tumor-infiltrating-lymphocyte (TIL) cultures and from healthy donors' peripheral-blood mononuclear cells (PBMC). MART-1/Melan-A27-35-specific CTL were generated from TIL after 2 weekly stimulation courses. Infection with rVV-M elicited a higher CTL response than addition of exogenous peptide, whereas, when a similar protocol was used to stimulate PBMC of healthy donors, significant and specific cytotoxic activity could be observed only upon rVV-M infection but not upon exogenous peptide addition. All CTL generated upon rVV-M stimulation were also able to efficiently kill melanoma cell lines expressing both MART-1/Melan-A and HLA-A2.1. In addition, TNF-alpha production could be induced in rVV-M-stimulated CTL upon co-culture with COS-7 cells transiently transfected with MART-1/Melan-A and HLA-A2.1 genes. This safe and highly immunogenic reagent could be of use in TAA-targeted clinical immunotherapy.