To define whether side-chain length influences the physiologic properties of bile acids, nor-ursodeoxycholate (nor-UDC), the C₂₃-nor derivative of ursodeoxycholate (UDC), was synthesized in both nonradioactive and radioactive forms (23-¹⁴C). Its hepatic translocation, hepatic biotransformation, and effect on bile flow, biliary bicarbonate, and biliary lipid secretion were compared with that of UDC and those of their respective glycine and taurine conjugates in anesthetized biliary fistula hamsters, rats, and guinea pigs, as well as the isolated perfused hamster liver. Hepatic uptake and biliary output of nor-UDC was slower than that of UDC or cholyltaurine in the isolated perfused hamster liver. In biliary fistula animals, nor-UDC was secreted only in bile. Biliary recovery of nor-UDC as compared to that of UDC was prolonged in the rat and hamster, although not in the guinea pig. Hepatic biotransformation, assessed by chromatography of bile, showed that conjugation of nor-UDC was inefficient, as unconjugated nor-UDC was present in bile; there was little amidation with glycine or taurine in any species, but sulfates and glucuronides, as well as other metabolites, were formed, with the pattern of biotransformation varying among species. When infused over a dosage range of 0.2–30 μmol/kg · min, nor-UDC induced a striking choleresis of canalicular origin. The bile acid-dependent flow was increased threefold in hamsters, ninefold in rats, and nearly twofold in guinea pigs when compared to that induced by UDC. The choleresis was associated with a linear increase in bicarbonate output and concentration in bile, and little phospholipid or cholesterol secretion was induced. A competition experiment in the bile fistula hamster indicated that nor-UDC or its metabolites, or both, appeared to compete for canalicular transport of ursocholyltaurine (a cholyltaurine epimer) when the latter was secreted under its V_max conditions. Conjugates of nor-UDC and UDC were promptly and almost completely recovered in bile without appreciable hepatic biotransformation; the conjugates did not induce a hypercholeresis or increase biliary bicarbonate concentration. It is proposed that a fraction of nor-UDC is secreted into canalicular bile in the unconjugated form and is protonated by a hydrogen ion derived from carbonic acid that was generated by the hydration of luminal CO₂ by carbonic anhydrase present in biliary ductular cells.