Biocatalytic reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate [(R)-EHB] with Acetobacter sp. CCTCC M209061 cells was successfully conducted in ionic liquid (IL) based biphasic systems. Several water immiscible ILs were used to construct biphasic, systems. The best IL investigated was 1-butyl-3-methylimidazolium hexafluorophosphate (C(4)mim center dot PF6), which also had good biocompatibility. Several influential variables were examined. The optimum parameters were as follows: volume ratio of buffer to C(4)mim center dot PF6, 1/2 (v/v); substrate concentration, 55 mmol/L; buffer pH, 5.5; cosubstrate concentration, 80 mmol/L; reaction temperature, 35 degrees C; and shaking speed, 220 rpm. Under these optimal conditions, the initial reaction rate, the yield, and the product e.e. were 0.39 mmol/(L min), 90.8%, and >99%, respectively, which were much better than results reported previously. This efficient whole cell biocatalytic process was feasible on a 450-mL preparative scale, and the immobilized. cells showed excellent operational stability and could be reused for at least 10 batches.