Horse, pig, and rabbit sera contain distinct glycoprotein inhibitors of influenza A viruses that inhibit hemagglutinating activity and neutralize viral infectivity. Although alpha 2-macroglobulin has been identified as the inhibitor in horse serum, the inhibitors in pig and rabbit sera have not been identified. As an initial step in elucidating the structural differences among inhibitor molecules, we sought to isolate the inhibitor in pig serum. The purified inhibitor decreased the hemagglutinating activity of influenza A virus, A/Los Angeles/2/87 (H3N2), and represented the majority of the virus-neutralizing activity in pig serum. The inhibitor corresponded in size to alpha 2-macroglobulin and cross-reacted antigenically with human alpha 2-macroglobulin. Characterization of the inhibitor's oligosaccharide moiety using linkage-specific lectins revealed the presence of N-acetylneuraminic acid-alpha 2,6-galactose but not N-acetylneuraminic acid-alpha 2,3-galactose. These data indicate that alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum.