A novel two-step acetone treatment method was developed to improve the enantioselectivity of Candida rugosa lipase (CRL) towards the hydrolysis of (R,S)-ketoprofen ethyl ester. After two-step acetone treatment of crude CRL, the CRL was harvested as precipitates and used for the production of optically pure (S)-ketoprofen. The specific activity of two-step acetone-treated CRL was 2.05 kU/mg protein, which corresponds to a 2-fold increase over that of crude CRL. The two-step acetone-treated CRL was considerably more enantioselective than the crude CRL towards (R,S)-ketoprofen ethyl ester, yielding an enantiomeric excess (% ee(p)) of about 100% and an enantiomeric ratio (E) of > 100. The hydrolysis activity of acetone-treated CRL increased with an increase in reaction temperature but the enantiomeric excess was > 99% regardless of reaction temperature. The production of optically pure (S)-ketoprofen was performed for 108 h in a scaled-up batch reactor containing 200 g of (R,S)-ketoprofen ethyl ester. Consequently, about 90 g of (S)-ketoprofen with an optical purity of 98% was recovered from the reaction mixture. (C) 2000 Elsevier Science Ltd. All rights reserved.