This report describes detailed protocols for the dissociation, seeding and growth in vitro of monolayer cultures derived from neonatal rat cerebrum. Primary cultures derived by using different seeding densities and in vitro ages were examined qualitatively and quantitatively for morphological composition in terms of two major cell classes (flat cells and process-bearing cells) and for the presence within these classes of glial fibrillary acidic protein (GFA) as detected by immunofluorescence histochemistry. Also examined was the reaction of the cells to serum withdrawal plus the administration of dibutyryl cyclic AMP in terms of the conversion of flat cells into process-bearing cells. Conditions are defined for the generation of in vitro cell populations, more than 90% of which are GFA-containing flat cells which can all be experimentally converted into cells with processes. These well-defined culture preparations will serve as useful models for future studies of astroglial behaviour.