The SSX-2 gene encodes a tumor-specific antigen expressed in neoplasms of various histological types. By analyzing a tumor-infiltrated lymph node of a melanoma patient bearing an SSX-2-expressing tumor, we have recently identified the first SSX-2-derived CD8⁺ T-cell epitope, that corresponds to peptide SSX-2_41–49, and is recognized by specific CTL in an HLA-A2 restricted fashion. Here, we have used fluorescent HLA-A2/SSX-2_41–49 peptide multimeric complexes to analyze the response to SSX-2_41–49 in melanoma patients and healthy donors. Multimer⁺ CD8⁺ T cells were readily detected in the majority of patients bearing SSX-2-expressing tumors and, at lower proportions, in patients with nonexpressing tumors and healthy donors. Importantly, isolated A2/SSX-2_41–49 multimer⁺ CD8⁺ T cells exhibited a large functional heterogeneity in terms of antigen recognition and tumor reactivity. SSX-2-specific CTLs isolated from tumor-infiltrated lymph node of antigen-expressing patients as well as from the corresponding peripheral blood mononuclear cells exhibited high functional avidity of antigen recognition and efficiently recognized antigen-expressing tumors. In contrast, SSX-2-specific CTLs isolated from patients with undetectable responses in the tumor-infiltrated lymph node, as well as from healthy donors, recognized the antigen with decreased functional avidity and were not tumor reactive. Together, these data indicate that CD8⁺ T-cell responses to SSX-2_41–49 frequently occur in SSX-2-expressing melanoma patients and suggest that SSX-2_41–49-specific CTLs of high avidity and tumor reactivity are selectively expanded during immune responses to SSX-2-expressing tumors in vivo.