Previous in vitro studies have shown that the oxidation of the side chain of bile acid precursors can start with either microsomal or mitochondrial enzyme systems. The microsomal system oxidizes the terminal methyl group (C-26) of the side chain that originates from C-2 of mevalonic acid, and the mitochondrial system oxidizes the terminal methyl group (C-27) derived from C'-3 of mevalonic acid. We administered [2-14C]-mevalonic acid to a patient with a complete bile fistula and isolated from the bile 3 alpha,7 alpha,12 alpha-trihydroxy[1,7,15,22,26-14C]5 beta-cholestanoic acid, a precursor in the synthesis of cholic acid which has undergone partial oxidation of the side chain. This compound was chemically decarboxylated and the liberated CO2 was trapped and counted. The recovery from control experiments using [24-14C]cholic acid was 82.6 +/- 2.3% (+/- 1 S.D.). However, following decarboxylation of 3 alpha,7 alpha,12 alpha-trihydroxy[14C5]5 beta-cholestanoic acid, only 7.5 +/- 0.58% (+/- S.D.) of the C-26 radioactivity was recovered. This study suggests that the major pathway of side chain oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol starts with hydroxylation of the methyl group derived from C'-3 of mevalonate (C-27) by mitochondrial enzymes. It is also concluded that the 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-27-oic acid formed in man has the R configuration at carbon 25.