Cytotoxic T lymphocytes (CTL) recognize class I major histocompatibility complex molecules complexed to peptides of eight to nine residues generated from cytosolic proteins. We find that CTL recognize, in vitro and in vivo, cells synthesizing a 10-residue peptide consisting of an initiating methionine followed by nine residues corresponding to a naturally processed determinant from influenza virus nucleoprotein (NP) (residues 147-155). Addition of two COOH-terminal residues corresponding to NP residues 157 and 158 severely reduced presentation of the endogenously produced peptide to CTL in vitro and in vivo. Extension of NH2 and COOH terminal flanking residues to include residues corresponding to NP residues 137-146 and 159-168 failed to increase the antigenicity of this peptide. Its presentation was greatly enhanced, however, by further extending the NH2 and COOH termini to include all of the additional residues of NP. These findings indicate first, that a naturally processed viral ligand (with an NH2-terminal Met) of a class I molecule contains sufficient information to access intracellular class I molecules, and second, that flanking residues can influence the processing and presentation of antigens to CTL.