Efficient photosynthetic energy transduction and its regulation depend on a precise supramolecular arrangement of the plant photosystem II (PSII) complex in grana membranes of chloroplasts. The topography of isolated photosystem II supercomplexes and the supramolecular organization of this complex in grana membrane preparations are visualized by high-resolution atomic force microscopy (AFM) in air in tapping mode with an active feedback control to minimize tip-sample interactions. Systematic comparison between topographic characteristics of the protrusions in atomic force microscopic images and well-established high-resolution and freeze-fracture electron microscopic data shows that the photosystem II organization can be properly imaged by AFM in air. Taking the protruding water-splitting apparatus as a topographic marker for PSII, its distribution and orientation in isolated grana membrane were analyzed. For the latter a new mathematical procedure was established, which revealed a preference for a parallel alignment of PSII that resembles the organization in highly ordered semicrystalline arrays. Furthermore, by analyzing the height of grana membrane stacks, we conclude that lumenal protrusions of adjacent photosystem II complexes in opposing membranes are displaced relative to each other. The functional consequences for lateral migration processes are discussed.