In anchorage-dependent (AD) cultures of the outer cell population (OCP) from neonatal rat calvaria, transforming growth factor-beta 1 (TGF-beta) specifically upregulated the synthesis of chondroitin sulfate (CS) proteoglycan (PG) and uncoupled the inhibitory effect of increasing cell density on CS PG synthesis (reference #30). Utilizing the same cell population, we have further examined the possibility that glycosaminoglycans (GAG) known to be synthesized and secreted by bone cells might exert feedback effects on GAG synthesis and/or its stimulation by TGF-beta. Although addition of TGF-beta alone stimulated net synthesis of HA and CS in both AD and anchorage-independent (AI) cultures, significant alterations of basal and TGF-beta-stimulated GAG synthesis by exogenous GAGs were observed only in AI cultures. In AI cultures exogenously added hyaluronic acid (HA) markedly enhanced the basal synthesis of HA and CS while heparin (H) suppressed the basal synthesis of HA, CS as well as dermatan sulfate (DS). Also, the addition of HA markedly potentiated the stimulation by TGF-beta of HA and CS synthesis as did heparan sulfate (HS) for CS and DS synthesis. H suppressed the stimulation of the synthesis of HA, CS and DS by TGF-beta. Overall, our results indicate specific effects of individual GAGs on basal and TGF-beta-stimulated GAG synthesis in OCP cultures. We suggest that some of the GAGs in the OCP microenvironment (which with the exception of HA are covalently linked to protein cores of secreted PGs), acting in concert with TGF-beta, may serve as an amplification system for upregulating GAG synthesis in the rapidly growing neonatal calvarium.