Cholic acid (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3 alpha,7 beta-dihyrdroxy-12-oxo-5 beta-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase and, the second by 7 beta-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with alpha-ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.