A simple assay was developed for the determination of glycine-conjugated bile acids. Samples containing the glycine-conjugated bile acids in the range from 15 to 250 nmol were acidified with HCl, and extracted with ethyl acetate containing ethanol (50 ml/l). An aliquot of the organic phase was evaporated to dryness, and the dried residue was treated to develop the color by the addition of acetic anhydride, pyridine and a trace amount of phosphoric acid. the absorbance was measured at 429 or 456 nm after the reaction at 50 degrees C for 2 h. Color development did not occur with unconjugated and taurine-conjugated bile acid. Beer's law was obeyed from 12.5 to 200 nmol in a cuvette. The recovery of the conjugates from the rabbit gall bladder bile and liver homogenate was satisfactory. This method requires no hydrolysis step and is applicable to the determination of glycine-conjugated bile acids in bile, duodenal aspirate and liver homogenate.