The substrate specificity of 3α-hydroxysteroid dehydrogenase (3α-HSD) toward the oxidation of the fetal bile acids, lβ-, 2β-, 4β-, 6α-, 6β-and 19-hydroxylated bile acids, was investigated using two kinds of commercially available kits for the enzymatic assay of total bile acids. The fetal bile acids having a hydroxy group at the 2β-, 4β-or 19-position were resistant to oxidation with 3α-HSD in both assay kits. However, a marked difference between the two assay kits in the oxidation of 1β-, 6α-and 6β-hydroxylated bile acids was observed. Therefore, the ability of 3α-HSD to oxidize the fetal bile acids would affect quantification of the total bile acids in biological fluids of newborns.