Equine alpha 2-macroglobulin (EM), known to contain both Neu5Ac and Neu4,5Ac2 sialic acid residues, was treated with Vibrio cholerae sialidase for the selective removal of Neu5Ac and was compared with the untreated EM for its binding by a panel of influenza viruses. Type A H3N2 virus strains having Leu in position 226 of their hemagglutinin (HA) changed the affinity for sialidase-treated EM only slightly, if at all, indicative of their ability to bind the 4-O-Ac-substituted Neu5Ac receptor determinant. At the same time, all B and H1N1 viruses, some H2N2 variants, as well as H3N2 strains with 226 Gln studied were unable to recognize Neu4,5Ac2 moieties of EM. Molecular modeling based on the known 3-D structure of H3 HA complexed with sialyllactose (Weis et al. (1988) Nature 333, 426-431) predicts that the 4-O-Ac substituent of sialic acid would protrude with its carbonyl oxygen inside the receptor-binding site of HA, thus possibly interfering with binding.