The partitioning of penicillin G acylase in aqueous two-phase systems (ATPS's) containing poly(ethylene glycol) (PEG) 20000 or 35000 and potassium dihydrogen phosphate (KH(2)PO(4)) or sodium citrate (C(6)H(5)Na(3)O(7)center dot 5H(2)O) has been measured at three temperatures, (301.2, 307.2, and 310.2) K. The effects of temperature, polymer Molecular weight, and polymer and salt concentrations oil the partitioning of penicillin G in the ATPS were Studied. The experimental data showed that the composition of salt has a large effect on partitioning of penicillin G in ATPS, and the temperature of the system has a small effect oil the partitioning. The UNIFAC-FV group Contribution model (Pazuki et it., Ind. Eng. Chem. Res. 2009, 48, 4109-4118) was used in correlating the partition coefficients of penicillin G in polymer + ATPS.