Transmission electron microscopy of freeze-etched and heavy-metal-decorated large protein complexes is capable of portraying their molecular symmetries and orientations. The technique in combination with image analysis has been applied to study the rotational order of individual lumazine synthase molecules either in crystals or adsorbed on solid substrates. On crystal surfaces rotational disorder was mainly observed for the molecules at and in the vicinity of relief perturbations. Molecules along surface steps and in the vicinity of vacancies showed no noticeable deviation from their translational and rotational order dictated by the lattice. Adsorption of lumazine synthase on mica led to layers with different degrees of coverage. Single, isolated molecules showed a slight preferential orientation with a hydrophobic region of their surface in contact with the substrate. In continuous, monomolecular adsorption layers microclusters have been observed in which the neighboring molecules were oriented in the same way with respect to the substrate. These microclusters may act as nuclei for the second layer which revealed a perfect crystalline order as do the consecutive layers. Thus, the multilayer structures grown on mica present real three-dimensional crystals except for the first layer contacting the substrate.