The partitioning and purification of polygalacturonases (PG) produced by Aspergillus niger URM 5162 were investigated in aqueous two-phase systems (ATPS), formed by polyethylene glycol and phosphate salts (PEG/phosphate). To evaluate the effect of the 4 independent variables molar mass of polyethylene glycol (PEG) (400-8000 g/mol - M-PEG), PEG concentration (12.5-17.5%, w/w - C-PEG), phosphate concentration (15-25%, w/w, C-PHOS) and pH (6.0-8.0) on the 4 response variables: partition coefficient (K), activity yield (Y), purification factor (PF) and selectivity (S), a factorial design (24) was used. The endopolygalacturonases (endo-PG) and exo-polygalacturonase (exo-PG) were preferentially partitioned in the top phase. For endo- and exo-PG, the highest values for the response variables K (1.23 and 2.40), Y (74.04% and 17.97%), PF (8.18 and 1.98) and S (24.68 and 48.07), respectively, were obtained for a C-PEG of 12.5% (w/w), M-PEG of 8000 g/mol, and C-PHOS of 25% (w/w) at pH 6.0. These conditions were considered the most suitable for the purification of PG produced by A. niger URM 5162. Furthermore, the most important independent variables for endo- and exo-PG were C-PHOS and M-PEG, respectively. All independent variables studied and their interactions significantly influenced the response variables. According to these results, the PEG/phosphate system is a useful cost-effective alternative for purification of PG produced by A. niger URM 5162. (C) 2014 Elsevier B.V. All rights reserved.