The partition and recovery behavior of phytase, produced by solid-state cultivation utilizing citrus pulp as substrate, was determined in an aqueous two-phase system (ATPS), composed by polyethylene glycol (PEG-citrate). The 2(4) factorial design was conducted to evaluate the effect of the following independent variables: PEG molar mass, PEG and citrate concentrations and pH, while partition coefficient, recovery and purification factor were selected as the variables responses. The best recovery in phytase activity (115.24%) condition was obtained with 26% (w/w) PEG 8000 (g/mol), 12% (w/w) sodium citrate at pH 8.0, showing a purification factor of 1.35. However, the highest partition coefficient (14.42) was observed within a 26% (w/w) PEG 400 (g/mol) and a 20% (w/w) sodium citrate at pH 6.0. The independent variables which more influenced on the partition coefficient and recovery were citrate concentration and PEG mass molar, respectively. The results suggest that PEG-citrate ATPS is an interesting and efficient alternative to partition and recovery of phytase from fermentation broth produced by Absidia blakesleeana URM5604. (c) 2012 Elsevier B.V. All rights reserved.