P revious studies have demonstrated that considerable amounts of parenterally administered cardiac glycosides are excreted in the bile and reabsorbed across the intestinal mucosa in several species. It is currently believed that the more prolonged action of nonpolar digitalis glycosides is due to their retention and recycling in the enterohepatic circulation. This report describes studies carried out to evaluate the effects of pharmacologic interruption of this enterohepatic cycle with the intraluminal sequestering agent cholestyramine.Cholestyramine was found to bind substantial quantities of digitoxin-³H and digoxin-³H in vitro and this binding was only modestly inhibited by the presence of bile. Administration of cholestyramine to rats by intragastric catheter before the subcutaneous injection of the LD₁₀₀ dose of digitoxin (10 mg/kg) resulted in a 70% survival rate. Further, oral administration of cholestyramine to rats before the subcutaneous injection of digitoxin-³H resulted in accelerated fecal excretion of radioactivity and lower levels of digitoxin-³H and metabolites in brain tissue compared to controls. Similarly, pretreatment of guinea pigs with cholestyramine orally before the injection of digitoxin in dosages of 10.0 and 4.0 mg/kg resulted in a 25 and 70% survival rate respectively as compared to survival rates of 0 and 30% in control animals. Cholestyramine pretreatment of guinea pigs was also accompanied by lower levels of digitoxin-³H and metabolites in heart and liver 90 min after injection of digitoxin-³H. Cholestyramine therapy did not result in significant changes in serum potassium levels excluding the possibility that drug-induced hyperkalemia might have affected the cardiac uptake of digitoxin.The data obtained in this study indicate that cholestyramine treatment affords a significant degree of protection against lethal digitoxin intoxication in rats and guinea pigs. It is suggested that cholestyramine binds appreciable amounts of digitoxin in the intestinal lumen resulting in reduced reabsorption, increased fecal excretion, and lower tissue levels of glycoside in critical organs. The protective effects of cholestyramine appear to be mediated by interruption of the enterohepatic circulation of digitoxin.