BACKGROUND: The development of efficient processes that will rapidly deliver new biological products to the market is a key goal in biochemical engineering. The establishment of purification processes that permit the recovery of closely related products is essential. The recovery and purification of these types of products is not a trivial task. The current approach exploiting chromatography techniques demands practical alternatives to address the technical and economic constraints. The potential application of countercurrent distribution in aqueous two-phase systems (CCD-ATPS) for the purification of related proteins is an alternative of great potential worth considering. RESULTS: The evaluation of the potential application of CCD-ATPS for the recovery of proteins provided conditions under which three model proteins (i.e. bovine serum albumin (BSA), ovalbumin and alpha-lactoalbumin) of similar isoelectric point but different molecular size can be separated. Process conditions PEG 1000 13% (w/w), phosphate 14% (w/w) at pH 7.0 and volume ratio of 1.0 resulted, after 32 transfers in CCD, in three distinguishable peaks that corresponded to each of the selected proteins. CONCLUSIONS: The findings reported here demonstrate a practical strategy that serves as a first step to establishing initial conditions to further exploit the separation of closely related proteins. (c) 2007 Society of Chemical Industry.