12-Keto-UDCA is an important optically active component for the drug ursodeoxycholic acid (UDCA). Starting from the three-keto compound dehydrocholic acid, the carbonyl groups at position 3 and 7 have to be reduced stereo- and regioselectively. In this case we applied two hydroxysteroid dehydrogenases for this purpose, the NAD-dependent 3α-HSDH from Pseudomonas testosteroni and the NADP-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Both enzymes can be produced in high yields by an Escherichia coli strain as recombinant proteins. In order to avoid impurities by the 7a-hydroxysteroid dehydrogenase of Escherichia coli, a mutant strain with an inactivated 7a-enzyme was applied for producing the three enzymes. For bioconversion, the dehydrogenases can be used as crude enzyme samples and are applied simultaneously. A 1.8 L batch of 100mM DHCA incubated at pH = 8.0 and 25°C resulted in 80 g crude product with a quite high purity of ≥ 99:5% as judged by HPLC analysis.