Ultrarapid freezing of bacteria (i.e. vitrification) results in optimal preservation of native structure. In this study, cryo-transmission electron microscopy of frozen-hydrated sections was used to gain insight into the organization of the Bacillus subtilis 168 cell envelope. A bipartite structure was seen above the plasma membrane consisting of a low-density 22 nm region above which a higher-density 33 nm region or outer wall zone (OWZ) resided. The interface between these two regions appeared to possess the most mass. In intact and in teichoic acid-extracted wall fragments, only a single region was seen but the mass distribution varied from being dense on the inside to less dense on the outside (i.e. similar to the OWZ). In plasmolysed cells, the inner wall zone (IWZ)'s thickness expanded in size but the OWZ's thickness remained constant. As the IWZ expanded it became filled with plasma membrane vesicles indicating that the IWZ had little substance and was empty of the wall's polymeric network of peptidoglycan and teichoic acid. Together these results strongly suggest that the inner zone actually represents a periplasmic space confined between the plasma membrane and the wall matrix and that the OWZ is the peptidoglycan-teichoic acid polymeric network of the wall.