Three-phase partitioning (TPP) was used to concentrate and purify alpha-galactosidase from watermelon (Citrullus vulgaris). The various process parameters required for efficient purification of alpha-galactosidase were optimized to get highest purity fold and yield. The best alpha-galactosidase yield (76.7%) with a nearly 2.7-fold purification was obtained in the interphase of the TPP system, which consisted of the crude extract to t-butanol ratio of 1:1 (v/v) in the presence of 50% (w/v) (NH4)(2)SO4 at pH 5.5. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a substantial level of purification of a-galactosidase from watermelon. The molecular weight of the enzyme was determined approximately as 45 kDa. The purified enzyme was characterized with respect to its activity and stability. Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and stability were studied. Optimum pH and temperature of a-galactosidase were determined as pH 6.0 and 60 degrees C, respectively. The purified enzyme was also very stable at a temperature range of 4-50 degrees C and a pH range of 4.0-6.5. The K-m and V-max, values were calculated from Lineweaver-Burk plot as 0.14 mM and 0.12 U, respectively. The results indicated that, TPP is a simple, quick, economical and very attractive process for primary purification of alpha-galactosidases compared to conventional chromatographic protocols. (C) 2013 Elsevier B.V. All rights reserved.