Six strains of lecithinase-lipase-negative Clostridia, isolated from human feces, were capable of oxidizing chenodeoxycholic acid to 3 alpha-hydroxy-7 keto-5 beta-cholanoic acid and of epimerizing it to ursodeoxycholic acid. The identity of the reaction products was confirmed by comparing their mass spectra, obtained by combined gas-liquid chromatography-mass spectrometry, with those of authentic reference compounds. 3 alpha-Hydroxy-7-keto-5 beta-cholanoic acid was reduced by growing cultures of all clostridial strains to chenodeoxycholic acid and to ursodeoxycholic acid, the latter being the preferred conversion product of most strains. However, ursodeoxycholic acid was not attacked by any of the strains. Growth kinetic experiments with three strains showed that chenodeoxycholate was transformed during the log or lag phase. No bile acid conversion could be seen during the stationary phase. While the concentration of chenodeoxycholic acid decreased and that of ursodeoxycholic acid increased tending towards plateaus, and concentration of 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid passes through a maximum. We proposed a reaction sequence with 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid as an intermediate for the epimerization of chenodeoxycholic acid to ursodeoxycholic acid. This demonstration is the first using isolated bacterial strains.