Fecal bile acid excretion was determined using recently developed techniques in order to investigate: the extent of the homogeneity in composition and concentration of individual bile acids in a single stool sample, the detailed qualitative and quantitative day-to-day variations in total and individual bile acids in the typical healthy adult, information on the relative proportions of conjugated bile acids in healthy stools, and inter-individual variations in fecal bile acid excretion. Bile acids were extracted from feces and separated into groups based upon their mode of conjugation using lipophilic gel chromatography, prior to analysis by capillary column gas chromatography-mass spectrometry. The majority of bile acids were excreted in the unconjugated form, while in all samples, conjugated bile acids accounted for less than 6% of the total fecal bile acids excreted, of which sulphated bile acids represented less than 3% of the total. Quantitative total and individual bile acid excretion, determined from single daily collections exhibited wide variations in values from day-to-day, and in accordance with early findings, indicates the need to use a minimum of 3- to 5-day collections for a more reliable index of bile acid excretion in feces. Examination of frozen and sectioned single stools revealed wide variations in water content and in quantitative bile acid concentration and composition within the stool. These data indicate random stool samples, which are commonly used in clinical studies, and data expressed as concentrations to be unsatisfactory for the accurate determination of fecal bile acid excretion.