Bicontinuous microemulsion was employed as the medium for enzymatic hydrolysis of (R,S)-ketoprofen ethyl ester in the presence of esterase for the first time. In addition, a methodology for the separation of optically pure ketoprofen from the microemulsion system for analysis by gas chromatography was developed. Various factors influencing the enzymatic hydrolysis of (R,S)-ketoprofen ethyl ester such as temperature, enzyme concentration and reaction time were optimized experimentally. The enzymatic hydrolysis in a bicontinuous microemulsion system showed a final conversion of 84.6% after 50 h of reaction, while hydrolysis in Tris-HCl buffer solution resulted in only 26.9% conversion after 150 h without completing the reaction. A comparison of the rate of the enzymatic hydrolysis reaction with rates of reaction in other biphasic media revealed that the bicontinuous microemulsion system was faster and more advantageous. The extremely large interfacial area of the latter fluid likely facilitated the contact between the catalyst and the substrate. Because the enzyme applied was not selective, formation of (R)-ketoprofen was also observed. Therefore, application of an enzyme with higher selectivity would provide better results. (C) 2010 Elsevier Ltd. All rights reserved.