Variability in growth characteristics of influenza B viruses remains a serious limitation in the manufacture of inactivated influenza vaccines. Currently, serial passage in eggs is the strategy used in most instances for selection of high growth virus variants. In previous studies we found that adaptation of the strain B/Victoria/504/2000 to high growth in eggs was associated with changes only in hemagglutinin (HA). The high growth phenotype was associated with acquisition of either two (R162M and D196Y) or three (G141E, R162M and D196Y) amino acid (AA) substitutions, predicted to be near the receptor-binding domain of HA. In the present study we analyzed, using reverse genetics, the contribution to virus growth of each of these AA substitutions and determined their effect on antigenic properties. We found that G141E and R162M were most favorable for virus growth; however, only R162M could improve virus growth without antigenic alteration. Substitution D196Y had least effect on virus growth but substantially altered antigenic properties. Additional virus variants with AA substitutions at positions 126, 129, 137 and 141 were generated and characterized. The AA changes advantageous for growth of B/Victoria/504/2000 were also tested in the context of the HA of the B/Beijing/184/93, a virus with stable low-growth phenotype. All of the tested AA substitutions improved the replicative capabilities of the corresponding viruses, but only N126D and K129E had no effect on antigenicity. The results of our studies demonstrate that introduction of specific AA substitutions into viral HA can improve viral replicative efficiency while preserving the original antigenic properties.