To investigate the molecular basis of the differential ability of human, equine, and guinea pig alpha 2-macroglobulins to inhibit hemagglutination and infectivity of a human influenza virus, A/Memphis/102/72 (H3N2), the structures of oligosaccharides released from the three glycoproteins by hydrazinolysis were analyzed comparatively. Approximately seven to eight sugar chains were released from each subunit of two potent inhibitors (equine and guinea pig alpha 2-macroglobulins) and a weak inhibitor (human alpha 2-macroglobulin). More than 70% of the oligosaccharides contained sialic acids in all three cases. Structural analysis of these sialo-oligosaccharides revealed that all of the three glycoproteins contain biantennary oligosaccharides with one and two sialic acids as major sugar chains (70-80% of total sugar chains). Four percent of the biantennary oligosaccharides from equine sample, 10% of those from guinea pig, and 24% of those from human contain a fucosylated trimannosyl core. No triantennary oligosaccharide was detected in equine alpha 2-macroglobulin. However, human and guinea pig alpha 2-macroglobulins contain both fucosylated and nonfucosylated triantennary oligosaccharides. All sialic acid residues occur as the Sia alpha 2----6Gal group. The one unique feature of the carbohydrate groups of equine and guinea pig alpha 2-macroglobulins was the presence of 4-O-Ac-Neu5Ac as 30-50% of the total sialic acids, while human alpha 2-macroglobulin contained only Neu 5Ac. However, 4-O-Ac-Neu5Ac is not responsible for the potent inhibition of influenza virus infection and hemagglutination as will be described in the accompanying paper.