Electron microscopy does not, in principle, require highly ordered crystals to determine a high-resolution structure. Nevertheless, crystals of any type help to constrain the molecules into a more limited range of orientations and positions, from which it is easier to carry out structure determination. We describe an improved procedure for determination of crystalline disorder, which we have applied to poorly ordered two-dimensional crystals of the chloride pump halorhodopsin from Halobacterium salinarum . The new image analysis procedure involves the use of a reference projection calculated from a global three-dimensional map to carry out the initial cross-correlation analysis. Coupled with a greater number of images taken with field emission gun microscopes, this has allowed us to calculate a three-dimensional structure for halorhodopsin, in which the seven transmembrane helices and certain molecular features, such as the β-ionone ring of retinal, are now resolved.