Fine specificity analysis of HLA B35-restricted Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) clones revealed a unique heterogeneity whereby one group of these clones cross-recognized an EBV epitope (YPLHEQHGM) on virus-infected cells expressing either HLA B*3501 or HLA B*3503, while another group cross-recognized this epitope in association with either HLA B*3502 or HLA B*3503. Peptide binding and titration studies ruled out the possibility that these differences were due to variation in the efficiency of peptide presentation by the HLA B35 alleles. Sequence analysis of the TCR genetic elements showed that these clonotypes either expressed BV12/AV3 or BV14/ADV17S1 heterodimers. Interestingly, CTL analysis with monosubstituted alanine mutants of the YPLHEQHGM epitope indicated that the BV12/AV3+ clones preferentially recognized residues towards the C terminus of the peptide, while the BV14/ADV17S1+ clones interacted with residues towards N terminus of the peptide. Molecular modelling of the MHC-peptide complexes suggests that the differences in two floor positions (114 and 116) of the HLA B35 alleles dictate different conformations of the peptide residues L3 and/or H7 and directly contribute in the discerning allele-specific immune recognition by the CTL clonotypes. These results provide evidence for a critical role for the selective interaction of the TCR with specific residues within the peptide epitope in the fine specificity of CTL recognition of allelic variants of an HLA molecule.