Eubacterium sp. strain VPI 12708 expresses inducible bile acid 7alpha-dehydroxylation activity via a multistep pathway. The genes encoding several of the inducible proteins involved in the pathway have been previously mapped to a bile acid-inducible (bai) operon in Eubacterium sp. strain VPI 12708. We now report the cloning, sequencing, and characterization of the baiG gene, which is part of the bai operon. The predicted amino acid sequence of the BaiG polypeptide shows significant homology to several membrane transport proteins, including sugar and antibiotic resistance transporters, which are members of the major facilitator superfamily. Hydrophilicity plots of BaiG show a high degree of similarity to class K and L TetA proteins from gram-positive bacteria, and, like these classes of TetA proteins, BaiG has 14 proposed transmembrane domains. The baiG gene was cloned into Escherichia coli and shown to confer an energy-dependent bile acid uptake activity. Primary bile acids were preferentially transported into E. coli cells expressing this gene, with at least sevenfold and fourfold increases in the uptake of cholic acid and chenodeoxycholic acid, respectively, over control reactions. Less transport activity was observed with cholylglycine, 7-oxocholic acid, and deoxycholic acid. The transport activity was inhibited by the proton ionophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and nigericin but not by the potassium ionophore valinomycin, suggesting that the transport is driven by the proton motive force across the cell membrane. In summary, we have cloned, sequenced, and expressed a bile acid-inducible bile acid transporter from Eubacterium sp. strain VPI 12708. To our knowledge, this is the first report of the cloning and expression of a gene encoding a procaryotic bile acid transporter.