The present paper deals with the proliferation and differentiation of glial cells in organotypic slice cultures of the rat hippocampal formation. Transverse slices of hippocampus of newborn to five-day-old rats were cultivated using the roller tube technique. To study the development of glial cells under these conditions, the slice cultures were processed for immunostaining employing antibodies against the glial fibrillary acidic protein. The proliferation of glial cells was studied in double-labeling experiments employing glial fibrillary acidic protein-immunostaining and the bromodeoxyuridine technique. The three-dimensional glial scaffold in the cultures was analysed in semithin and ultrathin cross-sections through the slice cultures after varying periods following explanation. Our results can be summarized as follows: 1. At all intervals after explanation of the slices there are numerous glial fibrillary acidic protein-positive cells with morphological characteristics of astrocytes. 2. With some modifications, the differentiation of astrocytes and their processes follows similar rules as observed in the hippocampus in vivo. A radial glial scaffold is also formed in the cultures. However, in cultures, a regular pattern of radial fibers is more obvious in the hippocampus proper than in the dentate gyrus. This glial scaffold persists after 20 days in vitro whereas it is known to disappear after the first postnatal week in vivo. 3. Bromodeoxyuridine-positive nuclei of glial cells were found at all time periods after explanation. After short incubation periods, they were most frequent in the "ventricular" zones of the cultures. Following longer incubation periods after bromodeoxyuridine administration, proliferating cells were found throughout the cultures, covering and underlying the cultured tissue. A rim of laterally migrating astrocytes completely surrounds the cultures. Our results demonstrate that glial cells proliferate and differentiate under the present culture conditions. After three weeks of incubation the whole slice culture is surrounded by a glial cover which may play an important role for the survival and differentiation of the cultured hippocampal neurons.