Poly(gamma-methyl L-glutamate)s with Ser, His, Asp, and Glu residues at the amino terminal as the serine protease catalytic site were prepared. The number-average degree of polymerization of the polypeptides was 51. A dipalmitoylphosphatidylcholine monolayer containing the polypeptides was formed at the air-water interface and was transferred onto gold-deposited glass plates, The binding of N-acetyltyrosine ethyl eater, a typical substrate of the serine protease, to the monolayer was characterized by surface plasmon resonance measurements. The four-polypeptide-lipid monolayer system conditioned on an aqueous solution containing the substrate N-acetyltyrosine ethyl eater exhibited Langmuir-type binding of the substrate, its binding constant of 6.1 x 10(4) M-1 was about 20 times larger than that observed for a monolayer prepared on pure water. The behavior may have arisen from a substrate-induced rearrangement of the four kinds of polypeptides in the monolayer, forming a substrate-binding structure similar to that found in serine protease.