Electron tomography of vitrified cells is a noninvasive three-dimensional imaging technique that opens up new vistas for exploring the supramolecular organization of the cytoplasm. We applied this technique to Dictyostelium cells, focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the cross-linking of individual microfilaments, their branching angles, and membrane attachment sites can be analyzed. At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26Sproteasome, can be identified in an unperturbed cellular environment.