Abstract The partitioning and resolution of two lipases (acidic and neutral) was achieved in an aqueous two-phase system from crude culture supernatant of Bacillus stearothermophilus SB-1. The effects of changes in PEG molecular weight, varying PEG/phosphate system, pH and sodium chloride concentration were examined on the partition coefficient, K in PEG/phosphate system. The best system was PEG 6000 (70% w/v)/phosphate (40% w/v) and NaCl (3% w/v) at pH 7.0 where the total lipase partitioned to the top PEG phase. The two lipases were then selectively eluted by addition of fresh salt phase in a pH dependent manner with acidic lipase moving to the bottom phase at pH 4.0 with 5.27-fold purification while neutral lipase shifted to the bottom phase at pH 6.0 with 15.25-fold purity. The two lipases were easily recovered from the salt phase by immobilisation on accurel. Thus the mixture of two lipases was resolved and purified into acidic and neutral within 2 h with complete recycling of both the phase components. It has also been found that addition of PEG 6000 leads to an increase in the enzyme activity of the lipase mixture.