Cryo-electron microscopy of frozen-hydrated thin sections permits the observation of the real distribution of mass in biological specimens allowing the native structure of bacteria to be seen, including the natural orientation of their surface layers. Here, we use this approach to study the fine ultrastructure of the division site, or septum, of Staphylococcus aureus D2C. Frozen-hydrated sections revealed a differentiated cell wall at the septum, showing two high-density regions sandwiched between three low-density zones. The two zones adjacent to the membrane appeared as an extension of the periplasmic space seen in this organism's cell envelope and showed no distinguishing structures within them. Immediately next to these were higher-density zones that corresponded to nascent cross walls of the septum. Unexpectedly, a rather broad low-density zone was seen separating cross walls in the septum. This mid-zone of low density appeared inflated and without visible structures in isolated cell walls, which showed only the high-density zones of the septum. Here, we suggest that frozen-hydrated thin sections have captured a highly fragile septal region, the mid-zone, which results from the dynamic action of autolysis and actively separates daughter cells during division. The two zones next to the membranes are periplasmic spaces. Immediately next to these are the growing cross walls composed of peptidoglycan, teichoic acid and protein.