Two hemagglutination inhibitors for influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 22, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the hemagglutination by influenza C virus strains but did not affect the hemagglutination by influenza A and B virus strains. Hemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with influenza C virus at 37 degrees C but not with the other types of influenza virus, indicating that the inhibitors are specific for influenza C virus. The inhibitory activity was also destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is cleavable by the former neuraminidase but not by the latter is essential for the hemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.