Flavin adenine dinucleotide (FAD) undergoes multisite molecular recognition at the air-water interface with mixed monolayers that are capable of complementary hydrogen bonding. Molecular images were obtained with an atomic force microscope (AFM) for mixed monolayers of guanidinium (G) and orotate (O) amphiphiles transferred from pure water and from aqueous FAD. The AFM image of the G/O mixed monolayer on FAD showed a periodic oblique pattern composed of two kinds of methyl peaks with different heights, whereas that on pure water showed a periodic hexagonal pattern of only one kind of methyl peak. The two-dimensional molecular patterning as height difference is conceivably caused by rearrangement of monolayer components based on specific recognition by the FAD template molecule.