The chromatographic support containing monolayers of phospholipids offers novel modes in analyzing and separating proteins. The polar choline head groups on immobilized phosphatidylcholine were used for the affinity purification of phospholipase A (PLA). The purification process involves removing the contaminating proteins with detergent additives to the elution buffer such as short-chain alkylsulfonates. The lipid-bound PLA was eluted with acetonitrile or octyllysophosphatidylcholine. The purity of PLA was approximately 70% based on densitometric scans of gel electrophoresis. These results suggest that the lipid-immobilized chromatography may be applied to develop purification methods for PLA, enzymes, and membrane proteins obtained from diverse cells.