Sialyl-linkage specificity of the sialidase of influenza B viruses isolated in different years from 1940 through 1990 (B/Lee/40,B/Setagaya/3/56,B/Tokyo/7/66,B/Kagoshima/1/68, B/Gifu/2/73, B/Kanagawa/3/76, B/Ibaraki/2/85, B/Yamagata/16/88, and B/Bangkok/163/90) was studied with N-acetylneuraminyl (alpha 2-3)- and (alpha 2-6)-lactoses, GM3 gangliosides containing the same sialyl-oligosaccharide sequences as sialyllactose, and also with type I and type II lacto-series gangliosides carrying Neu5Ac alpha 2-3Gal and NeuAc alpha 2-6Gal linkages as substrates. From an examination of up to nine strains, the sialidases of all viruses preferentially hydrolyze substrates with Neu5Ac alpha 2-3Gal linkage rather than the Neu5Ac alpha 2-6Gal linkage. It was found that the sialidase activity toward Neu5Ac alpha 2-6Gal linkage relative to Neu5Ac alpha 2-3Gal linkage is increased in later strains, whether sialyllactose or ganglioside is used as the substrate. These results suggested that the sialidase of influenza B virus isolates has shown a drift in linkage specificity which correlates with the year of isolation.