AbstractConjugated bile acids, namely glyco‐ and tauro‐3α,6α‐dihydroxy‐5β‐cholanoic acid (hyodeoxycholic acid), 3α,7α‐dihydroxy‐5β‐cholanoic acid (chenodeoxycholic acid), 3α,6α,7α‐trihydroxy‐5β‐cholanoic acid (hyocholic acid) and 3α‐hydroxy‐6‐oxo‐5β‐cholanoic acid (6‐keto‐litocholic acid) were isolated from pig bile, and subsequently transformed into the corresponding methyl esters. Separation of the methyl esters of the isolated bile acids by high‐performance liquid chromatography (HPLC) was accomplished on a ZORBAX‐CN column (Dupont, Boston, MA) withn‐hexane/2‐propanol/methylene chloride (89∶6∶5, by vol) as the mobile phase containing traces (≈1%) of amyl alcohol and water as moderators. HPLC analysis of the methyl esters also showed the presence of methyl 3α‐hydroxy‐6‐oxo‐5α‐cholanoate, which was probably produced in the course of alkaline hydrolysis of the conjugated bile acids.