The immobilization of phospholipase D produced by Streptomyces sp. YU100 was evaluated to see it would be practical for industrial applications. To accomplish this, the purified enzyme, which contained 53 unit/mg of protein, was subjected to immobilization on various matrices. When immobilization supports including calcium alginate gel, polyacrylamide gel, and macroporous resin were evaluated, the highest enzyme retention ratio (> 42%) was observed on a Dowex MSA-2 macroporous resin. This may have occurred as a result of the ability of the hydrophobic domain of phospholipase D to interact with the polystyrene backbone of the resin, as well as the ability of the dimethylethanolamine group of the MSA-2 resin to retain the enzyme by forming hydrogen bonds with the acidic residues of the enzyme. Upon the operation of a reactor packed with enzyme that had been immobilized on a Dowex MSA-2 resin, greater than 80% of the initial enzyme activity was retained for 16 days. During the reaction, phosphatidylcholine became bound to the immobilized resin and interfered with the enzyme reaction, therefore, the resin was washed with ethyl ether every 2 h. A process for recovering excessive L-serine from phospholipids using the Dowex MR-3 resin was designed, and the separated L-serine was employed again after replacing the amount that was used.