Bacillus subtilis neutral protease was produced, purified and partitioned in aqueous two-phase systems for determining isoelectric pH of the enzyme. The enzyme was purified through ammonium sulfate precipitation, DEAE-cellulose ion chromatography and CM-cellulose ion chromatography. DEAE- and CM-cellulose chromatography profiles showed that the protease was charged positively at neutral pH, unlike most proteins. Bacillus subtilis neutral protease was purified by a factor of 10.9 compared to the crude enzyme in the liquid culture broth. Cross partitioning with two different salts and various pHs using polyethylene glycol/Dextran aqueous two-phase systems was used for measuring the isoelectric points of model proteins (bovine serum albumin, conalbumin and lysozyme) and purified Bacillus subtilis neutral protease. The crossing points of bovine serum albumin, conalbumin, lysozyme and Bacillus subtilis neutral protease were pH 4.9, 6.2, 10.6 and 9.0, respectively. These results were the same as values of isoelectric pH of the corresponding proteins and the protease in the literature. It is concluded that the cross partitioning of two different salts can determine the isoelectric points of proteins accurately.