To define the recognition site of cytotoxic T lymphocytes (CTLs) on influenza virus H5 hemagglutinin (HA), an H5 HA-specific CTL clone was examined for the ability to recognize monoclonal antibody-selected HA variants of influenza virus A/Turkey/Ontario/7732/66 (H5N9). On the basis of 51Cr release assays with the variants, a CTL epitope was located near residue 168 of H5 HA. To define the epitope more precisely, a series of overlapping peptides corresponding to this region was synthesized and tested for CTL recognition. The minimum peptide recognized by the CTL clone encompassed residues 158 to 169 of H5 HA. Relative to the H3 HA three-dimensional structure, this CTL epitope is located near the distal tip of the HA molecule, also known as a major B-cell epitope on H3 HA. A single mutation at residue 168 (Lys to Glu) in the H5 HA variants abolished CTL recognition; this same amino acid was shown previously to be critical for B-cell recognition (M. Philpott, C. Hioe, M. Sheerar, and V. S. Hinshaw, J. Virol. 64:2941-2947, 1990). Additionally, mutations within this region of the HA molecule were associated with attenuation of the highly virulent A/Turkey/Ontario/7732/66 (H5N9) (M. Philpott, B. C. Easterday, and V.S. Hinshaw, J. Virol. 63:3453-3458, 1989). When tested for recognition of other H5 viruses, the CTL clone recognized the HA of A/Turkey/Ireland/1378/83 (H5N8) but not that of A/Chicken/Pennsylvania/1370/83 (H5N2), even though these viruses contain identical HA amino acid 158-to-169 sequences. These results suggest that differences outside the CTL epitope affected CTL recognition of the intact HA molecule. The H5 HA site defined in these studies is, therefore, important in both CTL and B-cell recognition, as well as the pathogenesis of the virus.