Two separation methods, aqueous two-phase (ATP) partitioning and cation-exchange chromatography, were compared as alternative methods for the recovery of recombinant dog gastric lipase (r-DGL) from extracts of transgenic corn endosperm. r-DGL is a hydrophobic, acid-stable protein targeted for stable expression in endosperm. Polyethylene glycol (PEG) - salt ATP system parameters of PEG molecular weight, phase-forming salt, NaCl addition, Triton X-100 concentration and phase ratio were adjusted to achieve favorable partitioning. The purification factor and yield of r-DGL in the bottom phase of a PEG 3350 (14.2%)-Na2SO4 (8.5%)-NaCl (0.5%)-Triton X-100 (2 mM) system at pH 4 were 1.5 and 80%, respectively. A higher purification factor of 2.3 and nearly 100% yield of r-DGL was obtained in the top phase of a PEG 3350 (9.4%)-phosphate (14.3%)-NaCI(1.5%)-Triton X-100 (2 mM) system at pH 4.0. The yield, purification factor, and concentration factor were 90%, 7.7, and 3.6, respectively, for the alternative of cation-exchange on CM-Sepharose. Countercurrent ATP partitioning with 3-7 stages was calculated to achieve a purification factor equivalent to that from cation exchange but with a lower concentration factor. While the cation exchange was favored on this basis, the two approaches were close enough that further optimization and economic analysis would be needed to be definitive.