β-Muricholic acid is the main bile acid in germ-free (axenic) rat but is found in smaller amounts in conventional (‘holoxenic’) rat. In order to study how intestinal microflora induces such a change in bile salt metabolism, the bacterial degradation of β-muricholic acid was studied in vivo and in vitro. Two conventional rats were fed a load of β-muricholic-4 14 C-acid and the distribution of radioactivity in the feces was studied by thin-layer chromatography with the following results: (1) β-muricholic-4 14 C-acid rapidly disappeared from the feces; (2) hyodeoxycholic acid was the main metabolite which was formed, together with 3α-hydroxy-6 keto-5β-cholanoic acid and several unidentified chemical species. However in vitro incubation of β-muricholic-4 14 C-acid with cecum homogenates of conventional rats did not result in the formation of hyodeoxycholic acid. The possible pathway of hyodeoxycholic acid formation in the rat is discussed.