Three-dimensional image analysis includes image processing, segmentation and visualization operations, which facilitate the interpretation of data. We have developed a toolbox for three-dimensional (3D) electron microscopy (EM) in Amira, which is a commercial software package, used by many laboratories. Our toolbox integrates a number of established procedures specifically tailored for 3D EM. These include input-output, filtering, segmentation, visualization and ray-tracing functions, which can be accessed directly from a user-friendly pop-up menu. They allow performing denoising and segmentation tasks directly in Amira, without the need of other programs, and ultimately allow the visualization of the results at photo-realistic quality with ray-tracing. They also allow a direct interaction with the data, such that, e.g., sub-tomograms can be directly extracted, or segmentation areas can be interactively selected. The implemented functions are fast, reliable and intuitive, yielding a comprehensive package for visualization in EM. (c) 2008 Elsevier Inc. All rights reserved.