Strategies to identify tumor-associated antigens rely on the paradigm that tumor-associated peptides presented in the context of HLA-class I are recognized by the cellular immune system. Approaches to isolate tumor-specific cytotoxic T lymphocytes (CTL) from tumor-infiltrating lymphocytes are difficult because long-term growth of the CTL requires autologous tumor cells and lymphocytes (PBL) as feeder cells. In this study, a CTL line (BL.HBL-100 CTL) was generated from PBL from a normal healthy donor by stimulating with irradiated, HLA-class I partially matched breast cancer cell line HBL-100. Activated T lymphocytes generated expressed TCRโฃ/โค ุ with a predominant CD8 ุ population after 12 stimulations (98.54% CD8 ุ vs. 0.18% CD4 ุ ). These CTL lysed HLA-A1 ุ , but not HLA-A1 ุ , breast cancer cell lines. Moreover, HLA-A1 ุ , non breast cancer cell lines were not recognized. The lytic activity of BL.HBL-100 CTL against HLA-A1 ุ breast cancer cell lines was blocked by monoclonal antibodies (MAbs) to HLA-class I and CD8, but not by anti-HLA-class II and CD4. Recognition of HLA-A1 ุ breast cancer cells by the CTL was dependent on peptides associated with HLA-class I since the lysis was inhibited by acid elution of HLA bound peptides. HBL-100 tumors were grown in severe combined immunodeficient (SCID) mice. Immunohistochemical staining of the HBL-100 tissue harvested from SCID mice demonstrated human breast cancer cells. HLAclass I molecules were affinity purified from the HBL-100 harvested from the SCID mice; class I bound peptides were eluted and separated by RP-HPLC. Pooled HPLC peptide fractions were tested for reconstituting antigenic epitopes recognized by the BL.HBL-100 CTL and found to reside within fraction 40. Our results show that a tumor reactive, HLA-class I restricted CTL was produced by stimulating normal PBL against an HLA-class I matched breast cancer cell line. We also provide evidence for a breast cancerassociated, HLA-class I bound peptide antigen(s) that reconstitutes the antigenic epitope(s) recognized by these CTL. Int.