The performance of a new NADP-regeneration system, based on the use of alcohol dehydrogenase (ADH)-acetone, has been investigated for the regioselective oxidation of cholic acid (1) to 12-ketochenodeoxy-cholic acid (2). Enzymes stabilities and substrate and/or product inhibitory effects under defined synthetic reaction conditions have been evaluated. The optimized system, based on a 4% w/v solution of I in a reaction mixture containing 25% v/v acetone, allowed the preparative scale transformation of 1 into 2 with a 92% conversion. (c) 2005 Wiley Periodicals, Inc.