The effect of poly(ethyleneglycol) (PEG) molecular weight, system pH, and sodium chloride concentration on the partitioning behavior of horseradish peroxidase from Armoracia rusticana root extract was investigated in poly(ethyleneglycol)/sodium phosphate systems. PEG molecular weight strongly affects the enzyme partition coefficient, whereas pH variation from 5.5 to 8.0 has little effect. The addition of sodium chloride (8% w/w) to a PEG 1540/phosphate system, pH 7.0, raises the peroxidase partition coefficient 13.5-fold without important changes in that of total horseradish root proteins. Moreover, these conditions allow direct homogenization of the A. rusticana roots with the selected aqueous two-phase system with the clear top phase containing over 90% of the enzyme and the purification factor being 4.8.