Partitioning of spleen proteinase from yellowfin tuna (Thunnus albacores) in an aqueous two-phase system (ATPS) was investigated. Phase compositions including PEG molecular mass and concentration as well as types and concentration of salts affected protein partitioning. ATPS comprising PEG1000 (15%, w/w) and magnesium sulfate (20%, w/w) provided the best condition for the maximum partitioning of the protemase into the top phase and gave a highest specific activity (47.0 units/mu g protein) and purification fold (6.61). The yield of 69.0% was obtained. Under the same ATPS condition used, the partitioning of protemase of splenic extract from three tuna species involving skipjack tuna, yellowfin tuna and tongol tuna were compared. The purity of splenic extract from all tuna species increased after ATPS process. Among all species tested, yellowfin tuna showed the highest purification fold, followed by tongol tuna and skipjack tuna, respectively. SDS-substrate gel electrophoresis revealed that the band intensity of major proteinase in ATPS fraction from all tuna species slightly increased with the concomitant decrease in band intensity of other contaminating proteins, indicating the greater specific activity of splenic extract. Therefore, ATPS was an effective method for partitioning and recovery of proteinases from tuna spleen. (c) 2005 Elsevier Ltd. All rights reserved.