The binding of influenza virus to erythrocytes and host cells is mediated by the interaction of the viral hemagglutinin (H) with cell surface receptors containing sialic acid (SA). The specificity of this interaction for 19 human and animal influenza isolates was examined using human erythrocytes enzymatically modified to contain cell surface sialyloligosaccharides with the sequence SA alpha 2,6Gal beta 1,4GlcNAc; SA alpha 2,3Gal beta 1,4(3)GlcNAc; SA alpha 2,3Gal beta 1,3GalNAc; or SA alpha 2,6GalNAc. Although none of the viruses agglutinated cells containing the SA alpha 2,6GalNAc linkage, differential agglutination of cells containing the other three sequences revealed at least three distinct receptor binding types. Several virus isolates exhibited marked receptor specificity, binding only to cells containing the SA alpha 2,6Gal or the SA alpha 2,3Gal linkage, while others bound equally well to cells containing either linkage. Moreover, some viruses could distinguish between two oligosaccharide receptor determinants containing the terminal SA alpha 2,3Gal linkage when present in the SA alpha 2,3Gal beta 1,4(3)GlcNAc sequence or the SA alpha 2,3Gal beta 1,3GalNAc sequence binding cells containing only the former. The observed receptor specificities were not significantly influenced by the viral neuraminidases as shown by the use of the potent neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. Receptor specificity appeared, to some extent, to be dependent on the species from which the virus was isolated. In particular, human isolates of the H3 serotype all agglutinated cells containing the SA alpha 2,6Gal linkage, but not cells bearing the SA alpha 2,3Gal beta 1,3GalNAc sequence. In contrast, antigenically similar (H3) isolates from avian and equine species preferentially bound erythrocytes containing the SA alpha 2,3Gal linkage. This is of particular interest in view of the identification of the avian virus H3 hemagglutinin as the progenitor of the H3 hemagglutinin present on the current human Hong Kong viruses.