A survey of 10 human influenza A viruses of the N2 serotype, isolated between 1957 and 1987, has revealed a drift in neuraminidase linkage specificity. While the earliest N2 strains examined exhibit strict specificity for cleavage of the NeuAcα2,3Gal sequence, N2 isolates from 1967 to 1968 also show limited activity towards the NeuAcα2,6Gal linkage. In strains isolated in 1972 and later, the N2 neuraminidase has approximately equal activity towards both types of linkages. The NeuAcα2,6Gal linkage cleaved by the later N2 neuraminidases is the preferred receptor determinant of human H2 and H3 hemagglutinins. Thus, the acquired neuraminidase specificity of the later isolates allows elution of bound virus from erythrocytes derivatized to contain the NeuAcα2,6Gal linkage, while earlier isolates, which cleave only the NeuAcα2,3Gal sequence, fail to elute from these erythrocytes. These results suggest that the observed drift in N2 neuraminidase specificity in the direction of the preferred H2 and H3 receptor determinant may facilitate release of progeny virus from host cells.