An attempt was made to study the partitioning behaviour of bovine serum albumin (BSA) in the PEG (polyethylene glycol)-sodium citrate based aqueous two-phase system. The investigation focus on the factors that influence partitioning of BSA, including PEG molar mass (4000, 6000 and 8000), and concentration, phase forming salt concentration, pH and temperature. The concentration of the BSA was measured by Bradford method. The partition of BSA to the salt rich (bottom) phase is strongly favoured in systems with higher molar mass of PEG, due to increase in hydrophobic nature of PEG with molar mass. The partition coefficient value tends to decrease with increase in PEG concentration, due to diminution of free volume available for the protein to accommodate in the top PEG rich phase. The enhanced affinity of BSA for the PEG phase increases with increase in sodium citrate concentration, due to an increase of the salting-out effect in the bottom phase. The results indicates that BSA partitioning equilibrium is displaced to the citrate rich-phase as the system pH increases, therefore the partition coefficient value changes depending upon the isoelectric point and protein charge. Furthermore, it is observed that the BSA partitions to the bottom phase at higher temperature. (C) 2010 Elsevier Ltd. All rights reserved.