The present review aims to give an overview of the cytochrome P450 8B (CYP8B) and cytochrome P450 4A (CYP4A) subfamilies in relation to biosynthesis of bile acids, in particular trihydroxy bile acids. Trihydroxy bile acids are basically required in most species and have an impact on cholesterol and lipid metabolism. The primary trihydroxy bile acid in most mammals is cholic acid. Some species produce other important trihydroxy bile acids, for example the adult pig which produce hyocholic acid instead of cholic acid. The position of the third hydroxyl group in cholic acid and hyocholic acid, 12α or 6α position, respectively, has a profound effect on the hydrophilic- hydrophobic property of the trihydroxy bile acids. The CYP8B subfamily is required for introduction of the 12α-hydroxyl group in cholic acid biosynthesis. The enzyme responsible for 6α-hydroxylation in hyocholic acid biosynthesis, however, varies among species. This review will discuss, in particular, porcine members of the CYP8B and CYP4A subfamilies because interesting findings regarding members of these subfamilies have recently been recognized in this species. CYP8B1 was for a long time believed to be absent in the pig but was recently found to be expressed in fetal pig liver. The enzyme catalyzing the 6α-hydroxylation in hyocholic acid biosynthesis in pig was found to be an atypical member of the CYP4A subfamily, denoted CYP4A21. The review presents bile acid biosynthesis in view of these findings and discusses physiochemical properties and developmental-dependent aspects related cholic acid and hyocholic acid biosynthesis.