As an effective extraction method, aqueous two-phase extraction systems based on imidazolium ionic liquids have been used, in this work, to extract proteins of bovine serum albumin, trypsin, cytochrome c and gamma-globulins. Effects of the ionic liquids concentration, pH value, and temperature of the systems on the extraction efficiencies have been investigated. Experimental results show that 75-100% of the proteins could be extracted into the ionic liquid-rich phase in a single-step extraction. The extraction efficiency of cytochrome c was changed slightly with the increase of pH values. Extraction efficiencies of the proteins were found to increase with increasing temperature and increasing alkyl chain length of cation of the ionic liquids. Thermodynamic studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting-out effects were also important for the transfer of the proteins. Importantly, conformation of the proteins was not affected after extraction into the upper ionic liquid-rich phase as determined by UV-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopy of the proteins. The enzyme activity of native trypsin and the trypsin in ionic liquids was determined using N-alpha-benzoyl-L-arginine ethyl ester as a substrate. This novel process is suggested to have important applications for the separation of proteins. (C) 2008 Elsevier B.V. All rights reserved.