Two bile acid extraction procedures were compared using endogenously radiolabeled tissues and feces. The method of Setchell et al. (J. Lipid Res. 24, 1085-1100, 1983) resulted in essential complete extraction, whereas that of Manes and Schneider (J. Lipid Res. 6, 376-377, 1971) gave recoveries between 56-82%. The time requirement for the method of Setchell et al. could be drastically reduced with no loss in extraction efficiency. Using extracts from endogenously labeled material, a purification procedure using C18 solid-phase extraction cartridges was developed that recovers >90% of bile acids. The distribution of bile acids within the intestinal tract and liver of the rat was determined.