The bile acids and bile acid sulphates in the urine, serum and bile of 8 cholestatic patients were studied quantitatively by gas liquid chromatography and gas liquid chromatography/mass spectrometry. The primary bile acids (cholic acid and chenodeoxycholic acid) comprised on average 94% of the total bile acids in bile, 70% in the serum and 64% in urine. The percentage composition of bile acids in bile was relatively constant and was not influenced by the degree of cholestasis. In contrast, in the serum only the primary bile acids were increased, the concentrations of the secondary bile acids (deoxycholic acid and lithocholic acid) and the minor bile acids remaining constant. The data do not support the hypothesis that monohydroxy bile acids accumulate in cholestasis and are related to the pathogenesis of this syndrome. The pattern of bile acid urinary excretion was similar to that in the serum. But in 1 patient, 3α,7β,12α trihydroxy 5β cholan 24 oic acid was a principal urinary bile acid, although very low concentrations of the compound were found in that patient's serum, suggesting that some of the minor bile acids in urine may originate by epimerization in the kidney. In bile only a small proportion of the bile acids was sulphated (range 2.1 to 4.6%) and in serum the degree of sulphation was very variable (9 to 50%). However, in urine, sulphate esters accounted for a large proportion of the total bile acids (33 to 72%). The output of bile acid sulphate in the urine was related to the urine total bile acid output but the serum concentration of bile acid sulphate remained relatively constant. Consequently, in contrast to the nonsulphated bile acids, whose renal clearance was relatively constant, the renal clearance of sulphated bile acids was directly related to the urine total bile acid output. This finding is inconsistent with the earlier hypothesis that their predominance in urine was due to a high renal clearance. It may indicate renal synthesis of some of the bile acid sulphates in the urine and/or inhibition of active renal tubular reabsorption of sulphated bile acids by nonsulphated bile acids.