Rosenthal fibers share epitopes with alpha B-crystallin, glial fibrillary acidic protein, and ubiquitin, but not with vimentin. Immunoelectron microscopy with colloidal gold. Ultrastructural immunoreactivities of alpha B-crystallin, glial fibrillary acidic protein (GFAP), ubiquitin, and vimentin in Rosenthal fibers (RFs) isolated from an Alexander's disease brain were investigated using nonosmium and low-temperature embedding technique. The morphology of RFs embedded in Lowicryl K4M resin was well preserved after treatment with 0.5% Triton X-100. alpha B-crystallin immunoreactivity was present in RFs of various sizes and was the strongest in loosely scattered deposits, which were considered to be the initial stage of RFs. Glial fibrillary acidic protein immunoreactivity in RFs was heavy, homogeneous throughout RFs, and equivalent to that in networks of glial filaments. Immunoreactivities of both alpha B-crystallin and GFAP were mainly restricted to the high electron-dense areas within RFs and were proved to exist close to each other by double immunolabeling. Rosenthal fibers were negative for vimentin. Ubiquitin immunoreactivity was relatively homogeneous in RFs with small diameters, but in RFs with large diameters, the immunoreactivity diminished in the center. Based on these observations, combined with the tendency of self-aggregation of alpha B-crystallin, it is conceivable that RFs are huge aggregation products of alpha B-crystallin involving GFAP, and that ubiquitination may be a consequent phenomenon, as it may be in other intracytoplasmic inclusions, such as neurofibrillary tangles and Lewy bodies. 