Previously, we detected a 43-kDa tumor-associated antigen (TAA) using the human monoclonal antibody L92, which recognizes the tetramer peptide KYQI. In the present study, cell lines of cytotoxic T lymphocytes (CTL) specific to the gp43 peptide (DLTMKYQIF) were established from peripheral blood lymphocytes (PBL) of melanoma patients. Patients' PBL (n = 326) of different HLA Class I types were assessed for gp43 CTL activity. CTL specific to gp43 peptide were generated only from HLA-A2 melanoma patients and not normal donors. gp43 CTL recognized gp43 peptide-pulsed autologous BLC and T2 HLA-A2 target cell lines. Furthermore, CTL lines were shown to kill both HLA-A2 autologous and HLA-A2 allogeneic melanoma cell lines, indicating that gp43 peptide can be processed endogenously and presented by melanoma cells as a common TAA. The gp43 CTL lines did not kill normal cells. Specific amino acids of the peptide were shown to be important determinants in stimulation and recognition of CTL. gp43 peptide, recognized by both antibodies and T cells of melanoma patients, is a novel TAA peptide that may play an important role in anti-tumor immunity in human.