Dendritic cells (DC) are potent stimulators of primary T cell responses. In this study, we demonstrate that DC, genetically engineered to express the MART-1/Melan-A (MART-1) tumor-associated Ag, express MART-1 mRNA and protein, correctly process and present the HLA-A2.1-restricted immunodominant MART-1 peptide (MART-1(27-35)), and serve as potent stimulators of MART-1-specific CTL in vitro. A replication-defective E1-deleted adenovirus (AdV) was constructed that expresses MART-1 (AdVMART1). Transduced DC produce full length MART-1 mRNA as well as MART-1 protein. AdVMART1 does not significantly down-regulate cell surface class I expression despite having an intact E3 region. Transduction of an HLA-A2-positive/MART-1-negative cell line with AdVMART1 renders these cells sensitive to lysis by CTL specific for the MART-1(27-35) immunodominant peptide. In addition, DC transduced with AdVMART1 stimulated MART-1(27-35)-specific tumor-infiltrating lymphocytes to synthesize IFN-gamma. Finally, AdVMART1-transduced DC were able to generate MART-1(27-35) peptide-specific, class I-restricted CTL in PBL cultures from normal donors. This study supports the use of tumor Ag-engineered DC in genetic immunotherapy.