A central composite rotatable design was employed to study the effect aqueous two-phase extraction conditions namely polyethylene glycol-1500 (PEG) concentration (12-16%), (NH4)(2)SO4 concentration (14-20%) and pH (6-9) on the extraction of luciferase from Photinus pyralis. Specific activity, activity recovery, purification fold, volume ratio, enzyme and protein partition coefficients were considered as response variables. The significant (p < 0.05) response surface models with high coefficients of determination values (R-2) ranging from 0.89 to 0.95 were fitted for the experimental data, which indicated that the polynomial response models fitted well for describing the specific activity, activity recovery, purification fold, volume ratio, enzyme and protein partition coefficients. Based on the design, the optimal conditions were polyethylene glycol concentration 12%, (NH4)(2)SO4 concentration ranging from 16.48% to 17.86% and pH ranging from 8.17 to 8.43. The graphical optimization of superimposed contour plots fulfilled the conditions to obtain specific activity (Y-1) >= 5.5 x 10(6) RLU/mg, activity recovery (Y-2) >= 70%, purification fold (Y-3) >= 6.5, volume ratio (Y-4) <= 0.77, enzyme partition coefficient, K-e, (Y-5) >= 95, protein partition coefficient, Kp (Y-6) <= 2.5. The study demonstrated that response surface methodology can be utilized for deriving the optimum conditions for optimizing the conditions for the aqueous two-phase extraction of luciferase. (C) 2013 Elsevier B.V. All rights reserved.