This investigation attempts to study and optimize the affinity partitioning conditions of papain in an aqueous two-phase system (ATPS). Reactive Red 120 was added to the ATPS as a free affinity dye ligand and five factors (pH, PEG concentration, ammonium sulfate, sodium chloride and dye) that influence the papain partitioning were analyzed using response surface methodology. The optimum conditions were determined as PEG 0.102 g/ml, ammonium sulfate 0.17 g/ml, sodium chloride 25 mg/ml, Reactive Red 120 4.5 mu g/ml and pH 8.0. It was observed that sodium chloride increased papain partitioning and Reactive Red 120 had little effect on partitioning. The optimal condition gave a K-U value for papain partitioning of 1.92 with a yield of 50.6%. (C) 2010 Elsevier B.V. All rights reserved.