A review of the 3 alpha-hydroxysteroid dehydrogenase method for determining the concentration of total bile acids in bile is described. The optimum conditions for the assay were established with respect to pH, temperature, incubation time, amount of NAD+, and units of enzyme activity required to obtain complete oxidation of the substrate under fixed conditions. Furthermore, the effect of hydrazine hydrate, methanol, and bile volume on the reaction was examined. It was also established that the bile acid concentration in bile samples with a high molar percentage of cholesterol would be overestimated if 3 beta-hydroxysteroid dehydrogenase were present with the 3 alpha-enzyme.