Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and maintained in culture. Proteoglycans were labeled with [35S]sulfate, [3H]glucosamine, or [3H]serine as precursors. Proteoglycans associated with the cell layer were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100. Labeled proteoglycans were isolated using sequential DEAE-Sephacel and Sepharose CL-4B chromatography under dissociative conditions in the presence of detergent. The cell layer contained three different hydrodynamic size species of dermatan sulfate proteoglycans, DS-Ic, DS-IIc, and DS-IIIc, and two different hydrodynamic size species of heparan sulfate proteoglycans, HS-Ic and HS-IIc, when analyzed on Sepharose CL-4B, DS-Ic, DS-IIc, and HS-Ic were indistinguishable from corresponding species in the medium fraction in terms of (a) hydrodynamic size analyzed on Sepharose CL-2B or Sepharose CL-4B, (b) buoyant density distribution in dissociative CsCl gradients, and (c) glycosaminoglycan and oligosaccharide compositions. The majority of these three proteoglycan species could be removed by brief trypsin treatment indicating their cell surface localization. Heparin released a part of the trypsin-removable proteoglycan population, which mostly consisted of DS-Ic with smaller amounts of DS-IIc and HS-Ic. Cell layer extracted with 4 M guanidine HCl without detergent contained a large hydrodynamic size structure excluded from Sepharose CL-2B, which contained DS-IIc and HS-Ic proteoglycans with large amounts of exogenous proteins. Proteoglycans were dissociated from this supramolecular structure in the presence of high concentrations of detergents (2% sodium dodecyl sulfate or Triton X-100), indicating the hydrophobic nature of this structure, probably a part of the plasma membrane, and suggesting that DS-IIc and HS-Ic are intercalated into membrane. Both DS-IIIc and HS-IIc were intracellular species, which were not released into the medium by the living cells or removed by either trypsin or heparin. DS-IIIc species was a single glycosaminoglycan chain having the same hydrodynamic size and sulfation pattern as those of glycosaminoglycan chains on DS-II. HS-IIc species was also a single glycosaminoglycan chain with an average molecular size only 1/3-1/4 that of chains on HS-I but heparan sulfate chains from both species showed a similar average sulfation pattern when analyzed by nitrous acid fragmentation. Both DS-IIIc and HS-IIc had small amounts of covalently attached peptides.