The important physiological role of bile acids in cholesterol homeostasis makes them an attractive target for drug interaction. Bile acids are synthesized from cholesterol in the liver, secreted into the small intestine, and recirculated back to the liver; interruption of this recirculation by bile acid sequestrants or surgery decreases blood cholesterol, but there is a demand for new nonsystemic hypocholesterolemics usable in small quantities. Inhibition of the Na+-dependent ileal bile acid transport system should reduce recirculating bile acids, and nonabsorbable specific inhibitors could be a new class of nonsystemic antiarteriosclerotic drugs. We report the first specific inhibitors of the ileal bile acid transport system, compounds 9-14, designed based on structural requirements for recognition by Na+-dependent bile acid transport systems (a negative charge in the side chain, at least one hydroxy group in positions 3, 7, or 12 of the ring system, and a 3-position modified with an additional moiety coupling two bile acid derivatives via a linker). Inhibition studies of Na+-dependent [3H]taurocholate uptake into rabbit ileal brush border membrane vesicles showed compounds 9, 11, and 12 had strong interaction with the transporter, stronger than cholic acid and comparable to taurochenodeoxycholic acid (TCDC). In situ ileal perfusion in anesthetized Wistar rats showed compound 12 exhibited strong inhibitory activity, while 9 and 11 had moderate activity. In conclusion, compounds 9, 11, and 12 represent the first specific inhibitors of ileal bile acid transporters, and extensive animal studies are underway to evaluate their pharmacological profile on bile acid and cholesterol metabolism.