An aqueous two-phase system of polyethylene glycol (PEG)–salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.