We report striking immunodominance in the neutralizing antibody responses of major histocompatibility complex congenic mice to natural infection with influenza virus (H3N2 subtype), as deduced by sequencing the hemagglutinin (HA) genes of monoclonal antibody (mAb)-selected mutant viruses. A majority of mAb, established from individual BALB/c (H-2d) mice, select mutant viruses containing the same single amino acid substitution in the membrane distal ectodomain, HA1 198 A→E, whereas changes at either HA1 158 G→E or HA1 198 A→E are selected for by mAb from BALB.K (H-2k) donors. The structural basis for immunodominance, and potential diversity of progenitor B cells, was investigated by sequence analysis of H and L chain gene rearrangements in mAb specific for HA1 158 or HA1 198. No correlation was found between antibody specificity and VH or VL gene usage, and a minimum of three to six progenitor cells contributed to the individual's repertoire for a single antigenic site. However, in a further analysis of the HA1 158-specific antibody response of CBA/Ca (H-2k) donors, there was highly restricted light chain gene usage. Focusing of the immune repertoire to limited regions of the HA molecule during a primary viral infection may be a significant factor in immune pressure for antigenic variation, particularly since there is no evident restriction in the antibody response to immunization.