Bile acid (BA) conjugation with the amino acids glycine and taurine ensures efficient solubilization, absorption, transport and excretion of Hydrophobic substances such as fat soluble vitamins and drugs in the gastrointestinal tract. The formation of BA conjugates is a two-step reaction, with the initial step cat alyzed by the membrane protein BAS, forming a BA C24 thioester bond with coenzyme A (CoA). Objective: In order to define the properties of BAS at the chemical, enzymatic and molecular levels, BAS was isolated from rat liver so as to characterize its kinetic properties in a purified state, and to prepare anti-rBAS antibodies. rBAS was purified 200-fold from rat liver using a com bination of Q-Sepharose, hydroxyapatite, and CM Sepharose chromatography. SDS-PAGE analysis of purified, reduced and denatured rBAS revealed that it is a 65 kDa protein (p65). In kinetic experiments, using purified rBAS, the Km for CoA was 130 ± 36 nM (30-fold lower than previous reports using crude microsomal preps) and the Km for chenodeoxycholic acid (CDC) was 2.13 ± 0.43 μM. The Vmax of rBAS was 206 ± 40 nmol min-1 mg protein-1. Using purified rBAS in liposomes to immunize mice, a panel of IgG monoclonal antibodies was produced, several of which showed immunoreactivity with human and rat liver BAS by ELISA and p65 by Western blot. Conclusion: Solubilization and purification of rBAS markedly altered its kinetic properties with CoA, either due to increased CoA availability or stability. The monoclonal anti-rBAS antibodies will provide valuble tools to further investigate bile acid conjugation.