Extraction of alkaline protease from cell free fermentation broth of Bacillus subtilis TISTR25 using aqueous two-phase systems of polyethylene glycol 1000 (PEG1000)-potassium phosphate to determine suitable extracting conditions (system pH, phase volume ratio, and fermentation broth loading) was studied in this work. System pH of 7.5 (at phase volume ratio of 1.0, and 20% (w/w) fermentation broth loading) was found most appropriate since high alkaline protease partition coefficient of 26.4, and yield of 96.3% were obtained, while achieving low total protein partition coefficient of 0.29. Phase volume ratio, in addition, was found to have a greater effect on alkaline protease partitioning and purity than fermentation broth loading. An aqueous two-phase system at pH 7.5 with phase volume ratio of 0.04 and 50% (w/w) fermentation broth gave high alkaline protease partition coefficient and the highest top phase purification factor of 41.2 and 6.1, respectively. However, low yield of 62.2% was determined. This indicated a balance between lower yield to higher product purity when extracting alkaline protease using a PEG1000-potassium phosphate aqueous two-phase system. (c) 2005 Elsevier Ltd. All rights reserved.