Secondary structural changes and partitioning process of bovine serum albumin (BSA) in the mixture of (poly(ethylene glycol 2000) + K2HPO4 + H2O) have been studied at 23 degrees C. The effect of varying weight percents of poly(ethylene glycol) and K2HPO4 on the secondary structure of partitioned BSA and the partitioning process of BSA were examined. The equilibrium weight percents of the obtained aqueous two-phase system (ATPS) have been evaluated, and the phase diagrams including the tie lines are presented. It was found that the magnitude of the partition coefficient of BSA and the secondary structures of partitioned BSA depend on the length of obtained tie lines. Occurrences of changes on the secondary structure content of the partitioned BSA were observed by FTIR spectroscopy investigation. The amounts of the secondary structure content of partitioned BSA as alpha-helix, beta-sheet, beta-turn, and unordered structures of BSA were determined and reported.