Betulinic acid, a triterpenoid found in many plant species, has attracted attention due to its important physiological and pharmacological properties. in order to obtain betulinic acid, betulin Was Submitted to transformation with the selected microorganisms. Betulin biotransformation was carried out with the filamentous fungi Armillaria luteo-virens Sacc QH (ALVS), Aspergillus foetidus ZU-G1 (AF) and Aspergillus oryzae (AO) under seven kinds of transformation condition. As a result Of transformation of betulin, A. luteo-virens Sacc QH was the best biocatalyst to produce betulinic acid under the designed conditions. Transformation caused by pre-cultured fungal mycelia, a process designated as G2. was favorable condition for betulin biotransformation as the productivity of betulinic acid was evaluated (>20%). M1 and M2 systems, where the betulin Substrate was micro-emulsified in mixtures of Tween 80 and organic solvents, were potential substitutes for G2. The possible pathway of betulin transformation is postulated in this work. The use of fungi and transformation mode described in current work are viable procedures for producing betulinic acid, which is of most importance to replace chemical synthesis ways. (C) 2009 Elsevier Inc. All rights reserved.