DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein-or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-α consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.