The central composite rotable design (CCRD) was used to determine optimal conditions for fibrinolytic enzyme production by Bacillus subtilis DC-2 in poly-ethylene glycol 4000 (PEG 4000) and sodium sulfate (Na2SO4) aqueous two-phase system (ATPS). PEG 4000 and Na2SO4 concentration, fermentation time and temperature, and pH were selected as variables to evaluate the fibrinolytic activity in PEG phase. Using response surface methodology (RSM), a second-order polynomial equation was obtained by multiple regression analysis. The predicted maximal fibrinolytic activity in PEG phase was 1241.02 IU/ml with 9.05% PEG 4000 concentration, 5.06% Na2SO4 concentration, 118.77 h fermentation time, 37.57 degrees C fermentation temperature and pH 6.52. The validity of the response model was verified by a good agreement between predicted and experimental results. The fibrinolytic activity obtained from experimental results in PEG phase (1223.61 IU/ml) was higher than that produced in homogeneous fermentation (1165.58 IU/ml). (C) 2007 Elsevier Ltd. All rights reserved.