The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383-391 SRYWAIRTR, was studied using HLA B27-restricted influenza-specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR V alpha 14 gene segment (but not those using TcR V alpha 12) were also sensitive to P1 substitutions, suggesting that the TcR alpha chain of these clones lies over the N terminus of bound peptide, and that the "footprint" of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule. These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are "flag" residues with TcR-accessible side chains.