Abstract Two-dimensional crystals of membrane-bound Na +,K +-ATPase were formed in acidic media and their qualities were investigated by electron cryo-microscopy as well as by conventional electron microscopy. At pH 4.8 in sodium citrate buffer, the best crystallization condition, more than 80% of membranes formed crystals. The high ratio allowed high-resolution images to be taken by electron cryo-microscopy. Image processing revealed that they had unique lattice constants ( a = 108.7 Å, b = 66.2 Å, γ = 104.2°) and had few defects in the crystalline arrays. The reconstituted Fourier map of the ice-embedded crystal showed that there are two high contrast parts in one unit cell.