We investigated the ability of HER-2 peptide E75, which maps an immunodominant CTL epitope for ovarian and breast tumor-associated lymphocytes (TAL), to activate effector functions in freshly isolated CD8+ cells from healthy individuals. IFN-gamma was rapidly induced by E75 within 20-24 h, in five of six healthy donors, in the presence of IL-12 and was detectable as early as 6 h. The IFN-gamma levels were Ag-concentration dependent. Similar results were obtained with peptides mapping CTL epitopes from two other tumor Ag: folate binding protein (FBP) and amino-enhancer of split of Notch (AES). IFN-gamma was also detected, from freshly isolated, unstimulated PBMC in response to HLA-A2 matched tumors + IL-12 but not of IL-12 alone. The major source of IFN-gamma were CD45RO+ CD8+ cells. Induction of IFN-gamma and IL-2 from CD8+ cells and of IL-12 from dendritic cells (DC) by CD8+ cells reactive with E75 mirrored their induction by the influenza matrix peptide (M1: 58-66) in the same individual. Responses to M1 are used to define the presence of activated memory cells in healthy individuals. Compared to M1 responses E75 recognition induced 2-4-fold lower levels of IL-12 from the same APC and IFN-gamma and IL-2 from the same CD8+ cells. At lower Ag concentrations the endogenous IL-12 induced by E75-reactive CD8+ cells did not reach the threshold required to co-stimulate for IFN-gamma. alphaB7.1 synergized with E75 in increasing the overall levels of IL-2 induced within 24 h. The presence of tumor Ag-reactive activated CD8+ cells in healthy individuals may improve our understanding of the mechanisms of immunosurveillance and regulation of immune responses by tumors.