The stereoselective phospholipase A2-catalyzed hydrolysis of patterned phospholipid bilayers consisting of the l- and d-isomers of α-dilauroylphosphatidylcholine (DLPC) and α-dipalmitoylphosphatidylcholine (DPPC) is reported. The stereochemically directed enzyme lithography demonstrated herein allows the parallel modification of large surface areas and constitutes a potentially useful method to structure biomimetic films, given the stereospecific action of many enzymes.