MHC peptides derived from tumor-associated antigens (TAAs) can serve as the basis for the development of immunotherapeutics to treat human malignancies. Previously, we identified novel HLA-A*0201 (HLA-A2)–restricted peptides recovered from soluble HLA molecules secreted by human tumor cell lines, transfected with truncated genes of HLA-A2 and HLA-B7. Here, 4 candidate peptides eluted from soluble HLA-A2 were selected on the basis of their precursor proteins being TAAs. Peptide p1028 (GLIEKNIEL), derived from DNA methyltransferase I (DNMT-1), which is overexpressed in various human tumors, showed the highest affinity to HLA-A2 and was relatively abundant in the sMHC/peptide complexes of all transfected breast, ovarian and prostate cancer cell lines. Peptide p1028-specific CTLs were generated in vitro and shown to efficiently lyse not only target cells pulsed with the peptide but also HLA-A2-positive breast cancer cell lines MDA-231 and MCF-7. The peptide induced IFN-γ production in CTLs, which were selectively stained by a p1028 tetramer. Since DNMT-1 is a widely expressed tumor-associated enzyme, the novel DNMT-1-derived, HLA-A2-restricted peptide GLIEKNIEL identified here may provide a suitable candidate for a therapeutic cancer vaccine. © 2004 Wiley-Liss, Inc.