Ganglioside GM1 was isolated from the small intestinal mucosa of man, pig, and beef and amounted to 0.1, 2.0, and 43 nmol per g fresh weight, respectively. These differences in GM1 content were associated with a quantitatively differing ability of the mucosal cells to bind cholera toxin. Human cells bound about 15,000 toxin molecules when saturated with the toxin, porcine cells 120,000, and bovine cells 2,600,000 molecules. The association constant (KA) of the cholera toxin binding was, for cells of all three species, about 10(9) liters/mol. Exogenously added GM1 ganglioside was incorporated in intestinal mucosal cells as well as in intact rabbit small bowel. The increment in GM1 was associated with a correspondingly increased number of binding sites for cholera toxin, whereas KA was unchanged. GM1 incorporation increased the sensitivity of the rabbit small bowel to the diarrheogenic action of cholera toxin. Vibrio cholerae sialidase hydrolyzed isolated intestinal diand trisialogangliosides to GM1. However, the enzyme did not change the ganglioside pattern of intestinal mucosa, had very little influence on the number of toxin binding sites on intestinal cells, and did not alter the sensitivity of the small bowel to the diarrheogenic action of the toxin. These results demonstrate a relationship in the intestinal mucosa between the GM1 ganglioside concentration, the number of binding sites for cholera toxin, and the sensitivity to the biologic action of the toxin. Thus, the study strongly supports the concept that the GM1 ganglioside is the intestinal binding receptor for cholera toxin.