The ability of Rhodococcus actinobacteria to transform betulin to betulone was proved and reported for the first time. Betulone, the product of regioselective oxidation of a 3 beta-hydroxyl group of betulin, is a useful intermediate in the synthesis of novel biologically active compounds. Of 56 strains of Rhodococcus tested, Rhodococcus rhodochrous IEGM 66 was selected because it had the highest betulin-transforming ability. It was shown that R. rhodochrous IEGM 66 growing cells transformed 0.5 g/L betulin to betulone with 45% conversion rate within 240 h. A substantial reduction in the time of betulin (0.5 g/L) biotransformation was achieved by using resting cells, which catalyzed the production of 75% betulone after 96 h. At higher initial betulin concentrations (1.0-3.0 g/L), resting cells catalyzed 40-60% betulone production within 24 h. (C) 2013 Elsevier Ltd. All rights reserved.