The Mollicutes (Mycoplasma, Acholeplasma, and Spiroplasma) are the smallest, simplest and most primitive free-living and self-replicating known cells. These bacteria have evolved from Clostridia by regressive evolution and genome reduction to the range of 5.8 x 10(5)-2.2 x 10(6) basepairs (bp). Structurally, the Mollicutes completely lack cell walls and are enveloped by only a cholesterol containing cell membrane. The Mollicutes contain what can be defined as a bacterial cytoskeleton. The Spiroplasmas are unique in having a well-defined, dynamic, helical cell geometry and a flat, monolayered, membrane-bound cytoskeleton, which follows, intracellularly, the shortest helical line on the cellular coil. By applying cryo-electron-microscopy to whole cells, isolated cytoskeletons and cytoskeletal fibrils and subunits, as well as by selective extraction of cellular components, we determined, at a resolution of approximately 25 A, the cellular and molecular organization of the cytoskeleton. The cytoskeleton is assembled from a 59 kDa protein. The 59 kDa protein, has an equivalent sphere diameter of approximately 50 A. Given the approximately 100 A axial and lateral spacings in the cytoskeletal ribbons and the near-circular shape of the subunit, we suggest that the subunit is a tetramer of 59 kDa monomers; the tetramers assemble further into flat fibrils, seven of which form a flat, monolayered, well-ordered ribbon. The cytoskeleton may function as a linear motor by differential and coordinated length-changes of the fibrils driven by conformational changes of the tetrameric subunits, the shape of which changes from near circular to elliptical. The cytoskeleton controls both the dynamic helical shape and the consequent motility of the cell. A stable cluster of proteins co-purifies with the cytoskeleton. These apparent membrane and membrane-associated proteins may function as anchor proteins.