We recently identified a tumor-associated antigen that was recognized by human monoclonal antibody L94. The antibody-reactive 707-AP sequence RVAALARDAP, cloned from a melanoma cDNA library, was also found to be recognized by peripheral blood lymphocytes (PBLs) from melanoma patients. In this study, 707-AP was used to stimulate melanoma patients' PBLs for the establishment of peptide-specific CTL cell lines. CTL cell lines derived from 258 melanoma patients of different human leukocyte antigen (HLA)-A and HLA-B allele expressions were assessed by a 51Cr cytotoxicity assay against the peptide-pulsed autologous B lymphoblastoid cells and T2 HLA-A2 antigen-presenting cells and autologous and allogeneic melanoma cell lines. The analysis of 707-AP CTL activity demonstrated that only HLA-A2 patients' PBLs could be stimulated with 707-AP. 707-AP CTLs were able to specifically lyse HLA-A2 autologous and allogeneic melanoma cell lines. This verified the endogenous processing and presentation of 707-AP by melanoma cells. 707-AP CTL cytotoxicity against peptide-pulsed autologous HLA-A2 B lymphoblastoid cells and T2 HLA-A2 cells was also demonstrated. The killing activity of HLA-A2 707-AP CTL cell lines (CD8+ CD3+) was inhibited by anti-HLA class and anti-HLA-A2 monoclonal antibodies. The amino acid substitution or deletion analysis of the 707-AP sequence in CTL stimulation and recognition confirmed that position 2, amino acid V and position 9, amino acid A were essential. Both positions are known as supermotif anchors for HLA-A2 peptide sequences. Our studies demonstrated that 707-AP is a potent stimulator of CTLs that can induce peptide-specific HLA-A2 melanoma cell killing. The recognition of 707-AP by both antibody and CTLs suggests its potential significance as a peptide immunotherapeutic.