In rat ovarian granulosa cell cultures, internalized cell surface heparan sulfate is processed by either a fast lysosome-mediated pathway or by a slow pathway which generates glycosaminoglycan fragments. Cell-associated dermatan sulfate proteoglycans also undergo processing through analogous pathways, although the slow pathway does not involve endoglycosidic cleavage of the dermatan sulfate chains. In the present study we tested whether intracellular glycosaminoglycan fragments in rat ovarian granulosa cells were transient residents of the nuclei. A technique for isolating nuclei was devised in which cells were lysed with a hypo-osmotic extraction buffer containing detergent. Nuclei were then purified by conventional methods, and final preparations gave excellent recovery of the starting DNA (approximately 90%). The technique was used to isolate glycosaminoglycans from nuclei after cells were metabolically radiolabeled with [35S]sulfate. The results indicated the possible presence of dermatan sulfate, but not heparan sulfate, glycosaminoglycans at this location. Failure to remove cell surface proteoglycans with trypsin before preparation of nuclei resulted in nuclear contamination with significant amounts of intact dermatan sulfate proteoglycans. Nuclei preparations obtained using traditional homogenization steps from cells either treated without or with trypsin gave identical results except that recoveries of DNA were much lower (approximately 30%). The results demonstrate the difficulty in isolating pure nuclei and therefore also of firm conclusions pertaining to the nuclear association of glycosaminoglycans.