To design a reliable 3D QSAR model of the intestinal Na ؉ /bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na ؉ /bile acid cotransporter. The IC 50 values of the training set of compounds covered a range of four orders of magnitude for inhibition of [ 3 H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na ؉ /bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na ؉ /bile acid cotransporter explain exactly the experimentally found structureactivity relationships for the interaction of bile acids with the ileal Na ؉ /bile acid cotransporter ( Kramer et al. 1999. J. Lipid. Res. 40: 1604-1617). The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a ) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b ) one of the ␣ -oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c ) the negatively charged side chain acts as hydrogen bond acceptor; and d ) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na ؉ / bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways. -Baringhaus, K-H., H. Matter, S. Stengelin, and W. Kramer. Substrate specificity of the ileal and the hepatic Na ؉ /bile acid cotransporters of the rabbit. II. A reliable 3D QSAR pharmacophore model for the ileal Na ؉ /bile acid cotransporter.