Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. Macromolecules are adsorbed onto a thin support film and a nanometer-sized electron probe is scanned across the specimen while the elastic-scattering signal is collected. The resulting digital image intensity is proportional to the local mass density of the specimen. Images can be recorded at low electron dose without significant radiation damage to the structures of interest. We have used this approach to determine the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri, which is protein assembly that produces motility in this simple bacterium. The structural unit appears to be a fibril, ~ 5 nm wide, composed of dimers of a 59 kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell.