Vero cells, MDCK cells and embryonated chicken eggs (eggs) were used to evaluate influenza virus growth characteristics and immunogenicity induced by inactivated influenza B vaccines. Both cell lines produced comparable quantities of total viral and haemagglutinin (HA) proteins. Sequence analysis indicated genetic identity of the HA of Vero- and MDCK-grown virus counterparts with maintenance of antigenic characteristics of viruses derived from humans. The egg-grown influenza B/Memphis/1/93 variant differed from cell-grown counterparts at amino acid position 198 (Pro-Thr) and lost a glycosylation site. The level of neuraminidase (NA) activity was the highest in egg-grown virus, while MDCK- and Vero cell-grown viruses possessed 70% and 90% less NA activity respectively when fetuin was used as a substrate. Although each of the vaccines induced high and comparable levels of serum antibodies, mammalian cell-derived vaccines induced antibodies that were more cross reactive, and those antibodies induced by egg-derived vaccine were more specific to the homologous antigen. ELISPOT analysis indicated that the mammalian cell-grown vaccines induced high frequencies of IgG-producing cells directed against both cell- and egg-grown antigens, while egg-grown vaccine induced high frequencies of IgG and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown virus antigen. Taken together, our results suggest that mammalian cells are a viable option for the production of influenza virus vaccines.