An optically pure (S)-naproxen was produced by two-step acetone-treated Candida rugosa lipase (CRL) through enantioselective hydrolysis of racemic naproxen methyl ester, The two-step acetone-treated CRL was much more enantioselective than the crude CRL towards the hydrolysis of (R,S)-naproxen methyl ester, yielding an enantiomeric excess (ee(p)) of >98% and an enantiomeric ratio (E) of >100. In terms of hydrolysis reaction rate and enantio selectivity, the optimal reaction conditions were found to be 37 degreesC and pH 6.0. The scaled-up production of (S)-naproxen was performed in a batch reactor containing 200 ml of substrate solution (50 mM MES buffer containing 200 mM (R,S)-naproxen methyl ester). After 156 h of reaction, 38.4% of the initial (R,S)-naproxen methyl ester was hydrolyzed, yielding an enantiomeric excess of 98% and an enantiomeric ratio of >100. Finally, (S)-naproxen was recovered from the reaction mixture with an optical purity of 98% and a recovery yield of 95%.