Three-dimensional electron microscopy of single macromolecular assemblies has made large strides forward over the last decade. A large number of image processing techniques have been developed and many have found general distribution. For the proper usage of the wide range of available techniques, a clear concept of all processing steps is essential. This chapter provides step-by-step instruction for the three-dimensional reconstruction of an unknown macromolecule. Where possible, the limitations of the techniques are explained. The chapter attempts to be sufficiently general such so as not to adhere to a single image processing system. Described are alignment techniques for two and three dimensions, classification procedures, and the usage of three-dimensional reconstruction algorithms.