Identification of naturally processed peptides recognized by tumor-specific CTLs may lead to epitope-specific tumor vaccines. Because these epitopes may be expressed differently on epithelial tumors and may differ in their ability to induce CTL in vivo, we have isolated the HLA-A2-peptide complexes by immunoaffinity from an established ovarian tumor line transfected with and expressing HLA-A2 gene. High-performance liquid chromatography-fractionated peptides were used to reconstitute epitopes recognized on HLA-A2 by three HLA-A2+ CD8+ CTL lines. These lines recognized at least three of the same groups of fractions (designated SKOV3.A, -B, and -C) but showed differences in the pattern of recognition of other fractions. To gain insight in the epitope distribution by freshly isolated ovarian tumors, we compared the recognition of peaks SKOV3.B and -C with the corresponding peaks from an ovarian tumor (OVA-6) that expressed similar levels of HLA-A2, using one of these lines (CTL-OVA-5) as indicator. CTL-OVA-5 recognized a large number of epitopes from peaks B and C rechromatographed on more resolving high-performance liquid chromatography gradients. Although a number of peaks appeared to be coincident on both SKOV3 and OVA-6, an even higher number appeared either not to overlap or to overlap only partially. These findings, which represent the first analysis of the epitopes presented by a patient tumor, suggest that the use of tumor line-derived peptides for vaccination may require selection of the epitopes corresponding to the ones presented by freshly isolated human tumors.