The unique properties of equine and guinea pig sera which make them potent inhibitors of influenza virus adsorption and infection have been investigated. The inhibitory activities of both sera are found to reside entirely in their respective alpha 2-macroglobulins, high molecular weight glycoproteins which bind to viral hemagglutinins via sialic acids of their N-linked carbohydrate groups. Structure analysis has shown that both proteins contain 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc) (Hanaoka, K., Pritchett, T. J., Takasaki, S., Kochibe, N., Sabesan, S., Paulson, J.C., and Kobata, A. (1989) J. Biol. Chem. 264, 9842-9849). These 4-O-acetylated sialic acids have been found in few species, making their coincidence with high inhibitory potency in equine and guinea pig alpha 2-macroglobulin striking. However, 4-O-Ac-NeuAc does not appear to increase the avidity of interaction with influenza virus since isolated oligosaccharides of equine alpha 2-macroglobulin are no more potent inhibitors of adsorption than isolated oligosaccharides of human alpha 2-macroglobulin, which is a relatively poor inhibitor and contains only NeuAc. Since 4-O-Ac-NeuAc is resistant to cleavage by viral sialidase it may serve to protect the inhibitor from inactivation. These and supporting results suggest that the key property of equine and guinea pig alpha 2-macroglobulin which make them high potency inhibitors is a spatial arrangement of sialic acid containing oligosaccharide groups which allows optimal interaction with multiple hemagglutinins. The implications of these results for the design of low molecular weight inhibitors of influenza virus infection are discussed.