A specific method for determination of cholic, chenodeoxycholic and deoxycholic acid in serum based on deuterium-labelled standards and selected ion monitoring is described. The coefficient of variation of the method as calculated from replicate determinations was in the range 1.4–3.3 % for all three bile acids. Recovery experiments were in accord with the high accuracy expected for this type of assay.In analyses of sera from 23 healthy subjects, the following concentrations were obtained in the fasting state: cholic acid, 0.29±0.81μmol/l; chenodeoxycholic acid, 0.18±0.32μmol/l; deoxycholic acid, 0.77±0.38μmol/l (mean±SD).The method may be suitable as a reference method and in experiments with a high need for accuracy.