In human liver homogenate the formation of bile acid-CoA thioesters is localized both to the microsomal fraction catalysed by an ATP-dependent synthetase and to the peroxisomal fraction catalysed by the thiolase in the last step of the beta-oxidative cleavage of the 5beta-cholestanoyl side chain. The cytosolic bile acid-CoA:amino acid N-acyltransferase catalyse the conjugation of the CoA-activated bile acids with taurine or glycine prior to secretion into bile. The formation of bile acid-CoA esters is considered the rate-limiting step in bile acid amidation. So far, a bile acid-CoA cleaving activity has not been assessed in the research of bile acid amidation in human liver. In this work, a bile acid-CoA cleaving activity has been demonstrated at a rate that may influence the concentration of bile acid-CoA thioesters, free bile acids and amidated bile acids within the hepatocyte. Recently, it was shown that free chenodeoxycholic acid, formed by the thioesterase, is the physiological ligand of the farnesoid X receptor. A multiorganelle distribution of the bile acid-CoA hydrolytic activity was found. In the postnuclear fraction of human liver homogenate, apparent Km and Vmax for the cleavage of choloyl-CoA were 7.7 x 10-5 mol/L and 3.6 nmol x mg-1 x min-1 respectively. The corresponding values for chenodeoxycholoyl-CoA cleavage were 7.1 x 10-5 mol/L and 4.8 nmol x mg-1 x min-1. Hydrolytic activities were detected in the microsomal and the peroxisomal fractions where the bile acid-CoA esters are formed as well as in cytosol housing the N-acyltransferase activity. Compared to the bile acid-CoA synthetase activities, the hydrolytic activities were considerably higher, both in the postnuclear fraction and in the microsomal fraction. The thioesterase activities were in the same range as detected for the N-acyltransferase activities both in the postnuclear fraction and in the cytosolic fraction. The mere presence of thioesterase in microsomes, peroxisomes and cytosol seems counterproductive to bile acid amidation. The thioesterases may have an indirect regulatory function on the bile acid synthesis and are important for the regulation of bile acid synthesis by providing free chenodeoxycholic acid, the most potent activator of the farnesoid X receptor.