With few exceptions, all bacteria possess a wall which protects them and controls their communication with the environment. In Gram-negative bacteria the cell wall exhibits a complex and unique multilayered organization. We have applied a modification of the freeze-fracture technique known as'fracture-flip' to visualize the real surfaces of the different wall layers in a Gram-negative bacterium, Escherichia coli. In combination with treatments to weaken the interlayer connections, this technique has provided new insights into the structure of the bacterial wall. Large areas of an intermediate layer (most probably the peptidoglycan-containing matrix) have been visualized for the first time between the plasma membrane and the outer membrane of the wall. Extensive regions corresponding to the cytoplasmic face of the plasma membrane have also been obtained. These images provide new three-dimensional views of the bacterial cell wall and provide the structural framework for the analysis of the molecular relationships between the different cell wall components.