Bile acids are converted to their glycine and taurine N-acyl amidates by enzymes in the liver in a two-step process. This conjugation reaction increases the aqueous solubility of bile acids, particularly in the acidic environment of the initial portion of the small intestine. In the first step, bile acid coenzyme A (CoA) thioesters are formed by a bile acid CoA ligase (BAL). This chapter describes the methods used to purify BAL from rat liver microsomes and to isolate and clone the cDNAs encoding BAL from a rat liver cDNA library, the expression of BAL, the assays used to measure its activities, and the chemical synthesis of bile acid CoA thioesters. Copyright 2005, Elsevier Inc. All rights reserved.