Glial cells in rat hippocampal slices cultured for 4 weeks were examined with immunocytochemical and cryoelectron microscopical methods. Astrocytes possessing long processes were similarly stained with antibodies against nestin, vimentin, and glial fibrillary acidic protein as seen by confocal microscopy. The three antibodies also labeled intermediate filaments in these astrocytes. In order to examine the fine structure of these intermediate filaments, slices were rapid-frozen for freeze-substitution and freeze-etching. By freeze-substitution the processes of the astrocytes were packed with large hundles of intermediate filaments. In rapid-freeze deep-etched slices, these filaments were often interconnected with filamentous cross-bridges. These cross-bridges were rather uniform in size and shape (mean 2.9 nm thick and 14.8 nm long). These results suggest that the filament network with these cross-linkers is important for shaping the long processes of nestin- and vimentin-immunoreactive astrocytes in slice cultures.