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Deuterium- and tritium-labeled chenodeoxycholic acid and lithocholic were prepared by catalytic reduction of their respective delta11 derivatives. Structures of the intermediates and their isotopic purity were verified by chemical ionization and electron impact mass spectrometry and by nuclear magnetic resonance spectroscopy. Experimental conditions for reductive deuteration were defined which gave complete reduction of the olefin and a product of high isotopic purity. Conditions for optimal tritiation were developed with which little exchange of protons with the solvent occurred; the product had high specific activity. To test biological stability of the label, the 3H-labeled chenodeoxycholic acid was administered simultaneously with 14C-labeled chenodeoxycholic acid to two healthy subjects and the 3H/14C ratio in bile was determined daily for several days. The ratio remained identical to that administered, suggesting that the 11,12-3Hlabel in chenodeoxycholic acid is stable during enterohepatic cycling and can be used for valid estimates fo bile acid kinetics in many by the isotope dilution technique.