Electron cryomicroscopy (cryo-EM) allows for the structural analysis of large protein complexes that may be difficult to study by other means. Frequently, maps of complexes from cryo-EM are obtained at resolutions between 10 and 25 angstrom. To aid in the interpretation of these medium- to low-resolution maps, they may be subdivided into three-dimensional segments representing subunits or subcomplexes. This division is often accomplished using a manual segmentation approach. While extremely useful, manual segmentation is subjective. We have developed a novel semi-interactive segmentation algorithm that can incorporate prior knowledge of subunit composition or structure without biasing the boundaries between subunits or subcomplexes. This algorithm has been characterized with experimental and simulated cryo-EM density maps at resolutions between 10 and 25 angstrom. (C) 2011 Elsevier Inc. All rights reserved.