We determined the amounts of [35S]-glycosaminoglycans (GAGs) found on the intracellular, pericellular and extracellular compartments of primary cultures of astrocytes derived from newborn rat cortex and cerebellum in vitro. Our results show that the greatest portion of newly synthesized GAGs were found in different cellular compartments, depending on the source of the astrocytes. In the cells derived from the cerebellum, the proportion of [35S]-GAGs secreted to the culture medium preponderates over the amount found in the two other compartments, whereas cells derived from the cortex accumulated higher proportions of [35S]-GAGs in the intracellular compartment than in the two other compartments. Cortical and cerebellar glial cells synthesised and secreted heparan sulfate (HS) and chondroitin 4-sulfate (C-4S). HS was predominantly accumulated on the pericellular surface, while C-4S was mostly secreted to the culture medium. Beside the difference on the distribution of total [35S]-GAGs among the three cellular compartments, no difference was observed on the relative proportions of HS and C-4S within each compartment. By defining the source of GAGs, the present study may help to complement and extend information on biosynthesis of these compounds by mammalian glial cells.