Active uptake of bile acids from the lumen of the small intestine is mediated by an ileal Na(+)-dependent bile acid transport system. To identify components of this transport system, an expression cloning strategy was employed to isolate a hamster ileal cDNA that exhibits bile acid transport activity. By Northern blot analysis, mRNA for the cloned transporter was readily detected in ileum and kidney but was absent from liver and proximal small intestine. The transporter cDNA encoded a 348-amino acid protein with seven potential transmembrane domains and three possible N-linked glycosylation sites. The amino acid sequence was 35% identical and 63% similar to the rat liver Na+/bile acid cotransporter. After transfection into COS cells, the hamster cDNA transported taurocholate in a strict Na(+)-dependent fashion with an apparent Km of 33 microM. This taurocholate transport was inhibited by various bile acids but not by taurine or other organic anions. The Na+ dependence, saturability, and bile acid specificity of transport as well as the tissue specificity of mRNA expression strongly argue that the transporter cDNA characterized in this study is the Na+/bile acid cotransporter described previously in ileum.