We examined whether immunization with a single peptide induces cytotoxic T lymphocytes (CTLs) of heterogeneous specificities in vivo. Immunization of BALB/c mice with the peptide H2:529-537, which corresponded to amino acid residues 529-537 on the HA2 subunit transmembrane region of influenza A/Jap virus (H2N2) and possessed an H-2Kd-binding motif, induced CD8+CD4- CTLs. These CTLs lysed influenza A/Jap virus-infected target cells as well as those pulsed with the H2:529-537 peptide. H2:529-537 peptide-induced CTLs also lysed to lower but significant levels the target cells pulsed with the H1:533-541 peptide, which corresponded to amino acid residues 533-541 on the HA2 subunit transmembrane region of influenza A/PR/8 virus (H1N1) and were compatible to H2:529-537. Immunization with the H1:533-541 peptide, which also possessed an H-2Kd-binding motif, induced CTLs in vivo. H1:533-541-induced CTLs lysed influenza A/PR/8 virus-infected target cells and those pulsed with the peptide H1:533-541. Subtype cross-reactive CTLs to the H2:529-537 peptide were not induced by immunization with the H1:533-541 peptide. Two peptides, H2:3S and H2:7S, which had one amino acid substitution, serine at the third and seventh positions, respectively, induced CTLs that lysed target cells pulsed with the respective peptides to the highest levels. These results indicate that immunization with a single peptide induces CTLs of heterogeneous specificities in vivo.