In this paper, the feasibility of applying the water-miscible alcohol-salt aqueous two-phase system to separate small molecular antibiotics in aqueous solution was proved. The partition behavior of tetracycline hydrochloride in the ethanol/2-propanol-(NH4)(2)SO4 aqueous two-phase system was investigated by determining partition coefficients and extraction efficiency. The partition coefficient and extraction efficiency of tetracycline hydrochloride in the ethanol-(NH4)(2)SO4 ATPS could reach 12.51% and 75.77% respectively at pH 2.00 and 0.50 g/mL (NH4)(2)SO4, furthermore, that of tetracycline hydrochloride in the 2-propanol-(NH4)(2)SO4 ATPS could reach 30.03% and 87.70% respectively at pH 1.52 and 0.50 g/mL (NH4)(2)SO4. The target was found to partition preferentially into the alcohol-rich phase in its cation form at pH<4.0. The effects of the concentration of salt, the type of alcohol, pH and temperature were discussed in detail. The partition of target to the top phase was enhanced by decreasing pH from its isoelectric point of 5.4 and increasing the concentration of salt and temperature. Another objective of this study was to discuss the underlying mechanism governing the uneven partition of target and phase-separation. The experimental phenomenon and results both indicate that the salting-out effect of salt is the driving force for the uneven partition of target, so the salting-out effect of salts on the separation of target cannot be neglected in discussing aqueous two-phase extraction mechanism. (C) 2010 Elsevier B.V. All rights reserved.