BALB/c mice immunized with a vaccinia virus recombinant expressing the influenza A virus (A/Udorn/72; subtype H3) hemagglutinin HA2 (H3HA2) induced a strong CD8+ cytotoxic T lymphocyte (CTL) response which was H-2d-restricted. Two peptides, derived from the primary sequence of the H3HA2 and consistent with the predictive motif for Kd-restricted epitopes, were tested for their ability to be presented to the CTLs by P815 cells. Peptides corresponding to the amino acids 93SYNAELLVAL102 and 181GYKDWILWI189 of the HA2 primary sequence sensitized target cells for lysis by the HA2-specific CTLs. Secondary in vitro stimulation with dendritic cells as a source of antigen presenting cells treated with peptide 93-102, or infected with recombinant vaccinia virus (HA2-VACC), induced type A cross-reactive CTLs from A/Udorn/72 immune spleen cells. This CD8+ CTL epitope overlaps with an H3HA2 I-Ad-restricted T helper epitope 96-104 recently reported by others. The H3HA2 epitope described here is the first CTL epitope in the influenza hemagglutinin which is found in all three subtypes of influenza A virus.