Abstract Electron microscope tomography produces three-dimensional reconstructions and has been used to image organelles both isolated and in situ, providing new insight into their structure and function. It is analogous to the various tomographies used in medical imaging. Compared with light microscopy, electron tomography offers an improvement in resolution of 30- to 80-fold and currently ranges from 3 to 8 nm, thus filling the gap between high-resolution structure determinations of isolated macromolecules and larger-scale studies on cells and tissues by light microscopy. Here, we provide an introduction to electron tomography and applications of the method in characterizing organelle architecture that also show its power for suggesting functional significance. Further improvements in labeling modalities, imaging tools, specimen preparation, and reconstruction algorithms promise to increase the quality and breadth of reconstructions by electron tomography and eventually to allow the mapping of the cellular proteomes onto detailed three-dimensional models of cellular structure.