Immunohistochemical techniques, done with fluorescein- and horseradish peroxidase-labeled antibodies specifically purified by immunoadsorption, were used to show cholera-toxin antigen in intestinal tissues from adult mice injected intraluminally with highly purified cholera toxin. The toxin (choleragen) and natural toxoid (choleragenoid), but not formalin-inactivated toxoids, were specifically and selectively adsorbed uniformly to the entire mucosal surface of the villi and crypt areas. No penetration of toxin into the epithelium or the lamina propria was observed. Ultrastructural localization studies, using the peroxidase-antibody method, revealed the toxin to be on the membranes of the microvilli. Autoradiography with highly purified, tritium-labeled toxin confirmed these observations. It is suggested that specific adsorption of toxin to the brush-border membrane is the initial step in the pathogenesis of cholera, but that an additional process, stimulated by toxin but not toxoid, is essential. This could be activation of adenyl cyclase. The possibility that amounts of toxin or toxin fragments below the limits of detection by the systems used could penetrate cannot be excluded. Since there is a systemic antitoxin response in cholera, this is likely to occur but may not actually be important in the pathogenesis of the diarrhea of cholera.