This study evaluated whether MF59-adjuvanted subunit trivalent influenza vaccine for the 2003/04 winter season (A/Moscow/10/99, H3N2; A/New Caledonia/20/99, H1N1; B/Hong Kong/330/01) would confer protection against mismatched and frequently co-circulating variants of influenza B/Victoria- and B/Yamagata-like virus strains. Haemagglutination inhibiting (HI) antibodies were measured in middle-aged and elderly volunteers against the homologous B/Victoria-like vaccine strain (B/Hong Kong/330/01) and against mismatched B/Victoria-like (B/Malaysia/2506/04) and B/Yamagata-like (B/Singapore/379/99 and B/Shanghai/361/02) strains. Immunization induced significant increases in the amounts of HI antibodies against all influenza B strains under investigation. However, the responses against the heterologous B/Shanghai/361/02 virus did not reach the desirable values of seroprotection. An age-dependent decline of the responses was found for B/Victoria-like antigens, but not for B/Yamagata-like strains. Although further studies are needed, our data support the recommendation of including influenza B viruses of the B/Victoria and B/Yamagata lineages in the future influenza vaccine preparations.