Genetic reassortment of the A/Shanghai/11/87 (H3N2) variant of influenza A virus with A/PR8/34 (H1N1) virus [the standard donor of high yield (hy) genes for influenza vaccine viruses] resulted in the isolation of two reassortants with differing H3 haemagglutinin (HA) phenotypes, X-99 and X-99a. The two HA phenotypes were derived from individual subpopulations of the H3N2 wild-type virus during the reassortment event. The HA mutants and their respectively derived reassortants (identical in RNA genotype) differed in antigenicity, replication characteristics, yield in chick embryos and haemagglutinin gene sequence. Despite antigenic differences in reactions to polyclonal rabbit antisera of 60%, both X-99 and X-99a, the hy reassortants, were equally immunogenic and protective in BALB/c mice to challenge by parental wild-type virus. Differences in HA phenotype were related to a Ser to Ile change at amino acid position 186. These findings emphasize the polymorphism of influenza virus strains as well as the need for caution in selection of vaccine strains from among antigenically distinct viral subpopulations.