The structure of the isolated cell envelope of Halobacterium halobium is studied by X-ray diffraction, electron microscopy, and biochemical analysis. The envelope consists of the cell membrane and two layers of protein outside. The outer layer of protein shows a regular arrangement of the protein or glycoprotein particles and is therefore identified as the cell wall. Just outside the cell membrane is a 20 A-thick layer of protein. It is a third structure in the envelope, the function of which may be distinct from that of the cell membrane and the cell wall. This inner layer of protein is separated from the outer protein layer by a 65 A-wide space which has an electron density very close to that of the suspending medium, and which can be etched after freeze-fracture. The space is tentatively identified as the periplasmic space. At NaCl concentrations below 2.0 M, both protein layers of the envelope disintegrate. Gel filtration and analytical ultracentrifugation of the soluble components from the two protein layers reveal two major bands of protein with apparent mol wt of approximately 16,000 and 21,000. At the same time, the cell membrane stays essentially intact as long as the Mg++ concentration is kept at treater than or equal to 20 mM. The cell membrane breaks into small fragments when treated with 0.1 M NaCl and EDTA, or with distilled water, and some soluble proteins, including flavins and cytochromes, are released. The cell membrane apparently has an asymmetric core of the lipid bilayer.