Information on the antigenic structure of influenza hemagglutinin (HA) has been deduced previously from sequence analyses of laboratory mutant viruses selected, in vitro, with neutralizing monoclonal antibody (mAb) established exclusively from BALB/c (H-2d) mice; and there has been no attempt to investigate the influence of host genetic background, or natural route of infection, on the protective antibody repertoire. CBA/Ca mice are extremely sensitive to X31 virus infection, and in the present study a structural analysis was made of the antibody repertoire, by direct sequencing of the HA genes of laboratory mutant viruses selected, in ovo with mAb from CBA/Ca mice primed by natural infection with X31 virus at two different infectious doses. Single nucleotide substitutions in the HA genes of mutant viruses identified both novel and immunodominant antigenic sites on the HA1 subunit: a majority of mAbs, from different donors, were of the IgG2a isotype and were specific for HA1 158 Gly. In addition, novel laboratory mutants were obtained containing substitutions in the HA1 subunit that had not been reported previously for H3 subtype viruses, either natural variants or laboratory mutants, at residues: HA1 62 Ile----Arg; HA1 165 Asn----Ser (resulting in the loss of a N-glycosylation site); and HA1 273 Pro----Leu. Our findings suggest that host genetic background and/or a natural route of infection may be significant factors in the selection of different and distinct neutralizing antibody responses to influenza HA and therefore be of some relevance in our further understanding of the immune pressure for antigenic drift, and the immunogenic features of a protective antigen.