Influenza H3N2 viruses have recently drifted from A/Wisconsin/67/05-like to A/Brisbane/10/07-like viruses. The effect of key amino acid substitutions in the hemagglutinin (HA) protein on the replication, antigenicity and immunogenicity of cold-adapted, live attenuated vaccine strains was examined. A/Brisbane/10/07 egg isolate contained a unique combination of G186 and P194 which were required for efficient virus growth in Madin–Darby Canine Kidney (MDCK) cells and embryonated chicken eggs, but the virus induced low level of serum antibody response in ferrets. Substitution of the G186 and P194 in the HA of A/Brisbane/10/07 by V186 and L194 that were present in the HA of A/Wisconsin/67/05-like viruses significantly impaired virus replication but greatly improved the immunogenicity of the vaccine virus to a level comparable to that elicited by the A/Wisconsin/67/05 vaccine. The replication of the variants with impaired growth could be improved by amino acid substitutions at the 195 or 226 residues. The viruses with the G186 and P194 residues were antigenically distinct from the viruses with V186 and L194. Sequence analysis of the HA sequences of the H3N2 viruses from the database and sequencing of the HA gene of a cell-derived A/Brisbane/10/07-like virus before and after egg passage indicated that the P194 residue was most likely derived from egg adaptation. Our results demonstrated the importance of careful evaluation of vaccine strains to ensure that the selected vaccines not only replicate well in eggs, but also retain their antigenicity and are immunogenic in the host.