BACKGROUND: The improved efficiency of steroid biotransformation using the biphasic system is generally attributed to the positive effect on the solubility of substrate in aqueous media. A promising alternative for the application of organic solvents in biphasic systems is the use of ionic liquids (ILs). This study aims to investigate the applicability of the biphasic ILs/water system for 11a hydroxylation of 16a, 17-epoxyprogesterone (HEP) by Aspergillus ochraceus. RESULTS: Of the seven ILs tested, [C3mim][PF6] exhibited the best biocompatibility, with markedly improved biotransformation efficiency. In the [C3mim][PF6]-based biphasic system, substrate conversion reached 90% under the condition in which buffer pH, volume ratio of buffer to ILs, cell concentration, and substrate concentration were 4.8, 10/1, 165 g L-1 and 20 g L-1, respectively. This is more efficient than that of the monophasic aqueous system. The effects of the cations and anions of these ILs on the 11a hydroxylation of 16a, 17-epoxyprogesterone (HEP) by A. ochraceus is also discussed. CONCLUSION: The above results showed that IL/water biphasic system improved the efficiency of 11a hydroxylation of 16a, 17-epoxyprogesterone (HEP) by A. ochraceus, thus suggesting the potential industrial application of ILs-based biphasic systems for steroid biotransformation. (c) 2012 Society of Chemical Industry