The partition behavior of pure alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, E.C. 3.2.1.1) in aqueous two-phase systems was examined in order to investigate the effects of changes in type of phase components on the partition coefficient K. Polyethylene glycol (PEG)/dextran, PEG/phosphate, and PEG/sulfate systems were evaluated. Factors such as PEG molecular weight (MW), pH, and concentration of NaCl were all found to influence K. At low values (<2 kUml(-1) phase system), enzyme concentration had no effect on K. At higher concentrations (up to 20.40 kUml(-1) phase system was used, corresponding to ca. 12.3 gl(-1), the phases became saturated and a twofold increase in K was observed, but also lower recovery. Subsequently, the separation and purification of alpha-amylase from typical contaminants from supernatant and whole both of Bacillus subtilis fermentation was examined. The best partition conditions were found in PEG 4000/phosphate systems with 8.8% w/w NaCl where the K can be increased 78 times from K = 1.3 to K = 100. These conditions were then used to study the effect of phase volume ratio (R) on the partition coefficient, the purification factor (PF), and recovery of alpha-amylase from industrial fermentations. R was found to influence the purification factor and the recovery, but not the partition coefficient. However, both alpha-amylase and contaminants partitioned to the top phase, leading to relatively poor separation (PF = 3.2 for R = 1). In a PEG/sulfate system, the addition of NaCl had an extreme effect on the partition behavior of alpha-amylase, giving an extreme K for alpha-amylase (K = 6,800) at a high concentration of NaCl (8.8% w/w) and an extremely low K (K < 0.005) at a lower concentration of NaCl. By exploiting this extreme partition behavior and manipulating R in a two-stage strategy, a 53-fold purification (with 86% w/w purity) could be calculated of a maximum possible of 63 at 100% w/w pure alpha-amylase.