Evidence is presented that peroxisomes are more important than other subcellular fractions in rat liver for the final reactions in the biosynthesis of cholic acid from cholesterol. The peroxisomal conversion of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid was studied in detail and optimal assay conditions were defined. It was shown that the reaction involves intermediary formation of 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid and that ATP, CoA, Mg++, NAD+ and FAD are necessary. With use of 18O2 and 2H2O it was further shown that the introduction of the 24-hydroxyl group in 3 alpha, 7 alpha, 12 alpha, 24 alpha-tetrahydroxy-5 beta-cholestanoic acid is the combined result of a desaturase and a hydratase. The reaction mechanism is thus analogous to that for beta-oxidation of fatty acids. The role of peroxisomes under conditions in vivo was studied in three patients with the rare inborn cerebro-hepato-renal syndrome of Zellweger. Apparently infants with this fatal disease have a complete lack of peroxisomes in the liver and kidneys. The patients were found to accumulate THCA and various polar metabolites of THCA in serum and bile. Administration of two 3H-labelled C27-precursors to bile acids (5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and 7 alpha-hydroxy-4-cholesten-3-one) resulted in a rapid conversion into THCA and a subsequent slow conversion into cholic acid. Administration of 3H-labelled THCA resulted in a slow conversion into cholic acid.(ABSTRACT TRUNCATED AT 250 WORDS)