The MAGE-3 gene is a member of a multigene family that is selectively expressed by subsets of different human tumor types, including malignant melanoma, but not by normal tissues except for testis and placenta. A cytolytic T lymphocyte (CTL)-defined MAGE-3 antigen, corresponding to the MAGE-3 peptide 271–279 associated with the human leukocyte antigen (HLA)-A2 molecule, has been recently identified using T lymphocytes from a normal individual stimulated in vitro with peptide-pulsed autologous antigen-presenting cells. Because MAGE-3 is expressed in 76% of metastatic melanomas, the HLA-A2-restricted MAGE-3 antigen should be expressed by approximately 37% of Caucasians bearing a metastatic melanoma tumor, thus representing an attractive candidate for the elicitation of specific CTL immune responses in vivo. In this study, we determined the proportion of HLA-A2⁺ melanoma patients displaying detectable MAGE-3 peptide 271–279-specific CTL precursors in peripheral blood. Peptide-specific CTL populations were obtained from at least 4 of 11 HLA-A2⁺ patients. Peptide-specific CTL lines derived from these populations readily lysed HLA-A2-positive target cells that were pulsed with MAGE-3 peptide 271–279 at nanomolar concentrations yet were unable to recognize (as assessed by cytolysis and cytokine production) MAGE-3-expressing autologous or allogeneic HLA-A2-positive melanoma lines. Similarly, the CTL lines failed to recognize MAGE-3-negative HLA-A2-positive tumor lines after transfection with the MAGE-3 gene, although they were able to recognize COS-7 cells transfected with MAGE-3. In contrast, HLA-A1-positive melanoma lines transfected with MAGE-3 were efficiently recognized by CTL lines directed against the MAGE-3 peptide 168–176, a known HLA-A1-restricted CTL epitope. These results suggest that the expression level of the MAGE-3 peptide 271–279, unlike that of MAGE-3 peptide 168–176, in melanomas may be too low to allow efficient recognition by specific CTLs. Thus, it appears that despite the presence of CTL precursors against MAGE-3 peptide 271–279 in some HLA-A2⁺ melanoma patients, the usefulness of this peptide for specific immunotherapy of melanoma may be limited.