The yeast strain CGMCC 0573 was identified as Citeromyces matriensis and shown to be capable of enantioselectively hydrolyzing ethyl ester of (R)-Ketoprofen (2-(3-benzoylphenyl)propionic acid). The strain was isolated for the first time from soil samples through a new and efficient screening procedure in which the probability of obtaining active strains was greatly increased by using ethanol and Tween-80 alternatively as additives during the enrichment culture. Studies of the culture conditions and catalytic performance of Citeromyces matriensis CGMCC 0573 showed that the enzyme occurs constitutively in the cells and its production is enhanced by feeding with Tween-80 during the early period of cultivation. Yeast extract was found to be beneficial both for growth and for esterase production. The optimal temperature and pH for the bioconversion were 40 degreesC and pH 8.0, respectively. Biotransformation using resting cells cultured in a flask with baffles and magnetic stirring and in the presence of 50 mM substrate resulted in the production of (R)-ketoprofen at 93% ee (enantiomeric excess) and at 42.6% conversion.