Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates, in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. We used the rAAV system to induce specific CTLs against tumor antigens for the development of ovarian cancer (OC) gene therapy. As an extension of the versatility of the rAAV system, we incorporated a self-antigen, Her-2/neu, which is expressed in many cancers, including breast and ovarian. We analyzed two different vectors containing a short (157-612) and long domain (1-1197). Our rAAV vector induced strong stimulation of CTLs directed against the self tumor antigen, Her-2/neu. We then investigated the efficiency of the CTLs in killing Her-2/neu-targeted cells. A significant MHC class I-restricted, anti-Her-2/neu-specific CTL killing was demonstrated against Her-2/neu-positive OC cells after one in vitro stimulation. In summary, single peripheral blood mononuclear cell (PBMC) stimulation with rAAV/157-612- or rAAV/1-1197-pulsed DCs induces strong antigen-specific CTL generation. The CTLs were capable of lysing low doses of peptides pulsed into target cells or OC Her-2/neu(+) tumors. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against OC antigens.