The peptide-binding and presentation characteristics of seven naturally occurring HLA-A2 subtypes were studied using M3(271), a peptide derived from the tumor-specific Ag encoded by gene MAGE-3, which has been shown to be processed and presented by A*0201+ melanoma lines. Three independent M3(271)-specific CTL clones were obtained from two unrelated A*0201+ donors. B lymphoblastoid cell lines (BLCLs) expressing A*0201, A*0207, or A*0209 could be sensitized to lysis by all three clones upon incubation with the relevant peptide. Furthermore, the same BLCLs were able to present endogenous M3(271) in IFN-gamma release assays. These findings demonstrate, for the first time, the existence of a functional overlap between A*0207 and other A*02 subtypes. One of the CTL clones also lysed M3(271)-pulsed BLCLs expressing A*0204 and A*0206, while the other two clones recognized M3(271) only in the context of either of these two subtypes. Peptide-pulsed BLCLs expressing A*0202 or A*0205 were not lysed, although A*0205 and, with lower affinity, A*0202 molecules were shown to bind peptide M3(271). These findings have implications for the selection of cancer patients for specific immunotherapy with peptide M3(271).