Mechanisms responsible for the difference in the relative amounts of taurine- and glycine-conjugated bile acid N-acyl amidates (Tau/Gly ratio) are not fully understood. In the present study, the stability of taurine- and glycine-conjugated bile acid N-acyl amidates during intestinal transit and absorption was examined to investigate the contribution of intestinal deconjugation to the Tau/Gly ratio in rat bile. Radiolabeled chenodeoxycholic acid (CDC) and its N-acyl amidates with glycine (CDC-Gly) or taurine (CDC-Tau) were introduced into the lumen of the upper small intestine in the biliary fistula rats, and radioactive metabolites in bile, blood, urine, and tissues were identified and quantitated by high-performance liquid chromatography. Results indicated that 1) extensive deconjugation of CDC-Gly occurs during intestinal absorption; 2) CDC-Tau is recovered in bile largely intact; and 3) newly synthesized CDC-Tau and CDC-Gly are formed in a ratio of less than 2:1 after administration of [14C]-CDC. In summary, the present study demonstrates that resistance of taurine-conjugated bile acid N-acyl amidates to hydrolysis in the intestine, rather than a difference in synthesis of taurine- and glycine-conjugated N-acyl amidates in liver, may account for the high Tau/Gly ratio in rat bile.