We describe for the first time the identification of 3α,7β-dihydroxy-5²-chol-22-en-24-oic acid (Δ22-UDCA) in the plasma, bile, intestinal contents, and liver tissue of Sprague-Dawley rats after intravenous and oral administration of ursodeoxycholic acid (UDCA). Infusion of [2,2,4,4-2H4]UDCA confirmed Δ22-UDCA to be a specific metabolite of UDCA. Definitive confirmation of this unique and major metabolite was established by liquid secondary ionization mass spectrometry and gas chromatography-mass spectrometry by comparison of the retention index and mass spectrum with an authentic standard of Δ22-UDCA. When rats were fed a diet containing 1.0% UDCA, high concentrations of Δ22-UDCA were found in the plasma (40.3 ±11.8 µmol/L) and liver tissue (300.9 ± 64.2 µmol/L) of tissue), and these represented 36% and 57%, respectively, of the UDCA concentration. In animals fed 0.4% and 1.0% UDCA, the mass of Δ22-UDCA in the jejunum was high (7.5 ± 0.9 and 6.6 ± 0.6 mg, respectively), accounting for 50-60% of the total UDCA, but diminished markedly along the intestine, accounting for <3% of the total UDCA in the colon. Although Δ22-UDCA was not found in biological samples from control rats, Δ22-β-muricholic and Δ22-ω-muricholic acids were normal constituents of plasma and intestinal contents and were major muricholate isomers in liver tissue and bile. Synthesis of Δ22-bile acids appears to be highly specific toward bile acids possessing a functional 7β-hydroxyl group. We presume that, in common with pathways for endogenous bile acid synthesis, partial side-chain oxidation of UDCA occurs in the peroxisome with formation of α/β unsaturation; since UDCA has only a 5-carbon side chain, release of propionic or acetic acid is not possible, β-oxidation proceeds no further, and Δ22-UDCA is formed. While the mechanism of formation and physiological significance of Δ22-bile acids remain to be established, our data indicate that partial β-oxidation is a quantitatively important pathway for endogenous bile acid synthesis and for UDCA metabolism in this species.