Attempts to detect a cytolytic T-lymphocyte (CTL) response in melanoma patients vaccinated with MAGE-3 peptides have been negative so far, even though some tumor regressions have been observed. The detection of such responses may require very sensitive detection assays for CTL precursors. To this end, we set up a method whereby a large number of CD8+ T-cell microcultures are stimulated with autologous antigen-presenting cells incubated with a peptide, in the presence of interleukin (IL)-6 and IL-12 during the first week, and IL-2 and IL-7 from the second week. We report here that not only monocyte-derived dendritic cells but also activated T cells incubated with the MAGE-3 antigenic peptide presented by HLA-A2 were effective in activating specific CTL precursors present in the blood of individuals without cancer. These precursors were detected in the CD8+ CD45RA+ subpopulation of T cells. Among the CD8+ T-lymphocyte population of blood donors, the frequency of CTL precursors specific for the MAGE-3.A2 antigen ranged from 4 to 17 x 10(-7). For the MAGE-3 antigenic peptide presented by HLA-A1, this frequency ranged from 0.4 to 3 x 10(-7). Knowing that several parameters of this procedure still have to be optimized, we will begin to use it to evaluate the CTL precursor frequencies of cancer patients before and after injection of MAGE peptides.