Remsen, C. C. (Swiss Federation Institute of Technology, Zurich, Switzerland), and D. G. Lundgren. Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques. J. Bacteriol. 92:1765-1771. 1966.-A comparison was made of the fine structure of the cell envelope of the gram-negative bacterium Ferrobacillus ferrooxidans when cells were prepared for microscopy by freeze-etching and chemical fixation techniques. Cell envelopes of chemically fixed cells appeared as five separate layers distinguishable by their location and electron density. Frozen-etched cells showed a three-layered complex with each layer measuring approximately 100 A in thickness. The latter technique is considered to be "artifact-free" and, as a technique, yields purely morphological information on the natural state. The three layers revealed by freeze-etching are: the outer layer, a lipoprotein-lipopolysaccharide layer; the middle layer, a layer composed of globular protein attached to fibrillar mucopeptide; and the innermost layer, the cytoplasmic membrane. The latter was covered with 100 to 120 A particles. The relationship of the aforementioned layers to those seen in chemically fixed cells is discussed.