The binding and hemagglutinating properties of cholera toxin (CT) were studied by competitive binding assays and hemagglutination inhibition. The binding of 125I-labeled CT to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1 among different inhibitors used. Other mono-, di-, and polysaccharides and glycoproteins were at least 105 times less potent inhibitors. On the other hand, hemagglutination of neuraminidase-treated human type B erythrocytes by CT was inhibited by lactose, galactose, hog A+H, bovine salivary mucin, porcine thyroglobulin, and fetuin, whereas that was not effectively inhibited by ganglioside GM1 at the highest concentration. These findings suggest that the predominant binding substance for CT on human type B erythrocytes is ganglioside GM1 and that hemagglutination requires some additonal process since the interaction of CT with ganglioside GM1 is somehow different in hemagglutination.