2164U90, [(3R,5R)-trans-3-butyl-3-ethyl-2,3,4,5-tetrahydro-5-phenyl-1,4- benzothiazepine 1,1-dioxide], was found to be a potent inhibitor of the ileal bile acid active transport system. In vitro, 2164U90 decreased uptake and active transport of taurocholic acid by rat everted ileal sacs with IC50s of 4.0 microM and 1.5 microM, respectively. In vivo, 2164U90 produced dose-dependent increases in 23,25-75Se-labeled homocholic acid taurine (SeHCAT) fecal excretion in rats and mice at doses of 3-30 mg/kg and 1-10 mg/kg, respectively. In rats, 30 mg/kg 2164U90 was equivalent to 500 mg/kg cholestyramine. Two days oral administration of 10 mg/kg 2164U90 to rats decreased the bile concentrations of total bile acids 42%, orally administered [3H]taurocholic acid ([3H]TC) 82%, and cholesterol 35%. Cholestyramine (500 mg/kg) had effects similar to 2164U90 on total bile acid and orally administered [3H]TC concentrations but had no effect on biliary cholesterol. The hypocholesterolemic activity of 2164U90 was determined in cholesterol-cholic acid-fed rats and cholesterol-cholic acid-coconut oil-fed mice. 2164U90 inhibited the dietary-induced increase in dextran sulfate-precipitable lipoprotein cholesterol (VLDL+LDL) at doses comparable to doses needed to increase the fecal excretion of bile acids. These data indicate that 2164U90 decreases bile acid absorption by inhibiting the ileal bile acid active transport system, resulting in hypocholesterolemic activity.