Purple membrane (PM) fragments spread over an air-water interface show both preferential orientation and reverse orientation (i.e., the cytoplasmic side of bacteriorhodopsin being oriented toward the water and toward the air, respectively). We used a spectroscopic surface denaturation quantifying technique and a current-measurement technique to evaluate the ratio of the amount of preferentially oriented bacteriorhodopsin (BR) to the amount of all BR in a PM film. This ratio decreased as the initial coverage (the ratio of the total area of all the PM fragments spread on the air-water interface to the area of the air-water interface before the PM was spread over the interface) increased, when the initial coverage was high (more than 0.3). We concluded that this decrease was due to the limited rotational diffusion caused by frictional interaction among PM fragments in a highly concentrated PM solution spread over a water surface.