The presence of tumor-reactive CTLs in tumor infiltrates and in the peripheral blood of cancer patients demonstrates an immune response against tumors that apparently cannot control disease spread. This raises concerns as to whether amplification of this response may be useful during disease progression. Induction of tumor-reactive CTLs in healthy donors at risk, as well as in patients free of disease, may be therapeutically important, based on the hypothesis that CTLs that recognize tumors early may be more effective in containing their progression than CTLs that expand only when the disease progresses. To address the feasibility of priming cytolytic activity in healthy donors, we used the HER-2 peptide E75 (369-377) as an immunogen and autologous peripheral blood mononuclear cell-derived dendritic cells as antigen-presenting cells. We found that of 10 healthy donors tested, two responded at priming with E75 presented on autologous dendritic cells by induction of E75-specific CTL activity. Three other responders were identified after two additional restimulations. Of these five responders, three recognized E75 presented on the ovarian tumor line SKOV3.A2, as demonstrated by cold-target inhibition experiments. Induction of cytolytic activity at priming was enhanced in responders by tumor necrosis factor-alpha and interleukin 12 but not in the nonresponders. AlphaB7.1 monoclonal antibody added at priming enhanced induction of lytic activity in only one of the four nonresponding donors tested, suggesting that in the majority of donors, E75-precursor CTLs were not tolerized. Because of the possibility that disease may develop in nonresponders, strategies to improve the immunogenicity of tumor antigens for healthy donors may be required for development of cancer vaccines.