The composition of the subphase to obtain a stable organophosphorus hydrolase (OPH) monolayer was reexamined. The surface pressure-area (g-A) isotherms show that a subphase at pH 7.6 with 0.5 M KCl can lead to a limiting molecular area close to the X-ray crystal data. The stability of the OPH monolayer at the air-water interface was tested by two different methods: the compression-decompression cycle isotherms and kinetics measurements. There was no change in the limiting molecular area during the compression-decompression cycle isotherms and only a very small change over a period of 120 min at constant surface pressure of 20 mN m(-1) was observed, which are good indications for a stable OPH monolayer formation at the air-water interface. The surface potential-area isotherm was measured for OPH at the air-water interface, indicating the orientation of the polar moiety of the macromolecules during the compression. The UV-vis absorption spectra of the monolayer were recorded in situ at the air-water interface to investigate the organization of the OPH molecules. Topographies of OPH molecules at the air-water and the air-solid interfaces were studied by Brewster angle microscopy (BAM) and atomic force microscopy (AFM), respectively. The size of the molecules measured from the AFM image agrees with the limiting molecular area per molecule.