L cells and endothelial cells synthesize a heparan sulfate (HS) subpopulation, HSact, that exhibits anticoagulant activity due to a specific monosaccharide sequence; the remaining heparan sulfate, HSinact, lacks this region of defined structure and is anticoagulantly inactive. HSact biosynthesis was examined in these two cell types by stably expressing epitope-tagged rat ryudocan (ryudocan12CA5), which possesses three glycosaminoglycan (GAG) acceptor sites. Both HSact and HSinact were present on ryudocan12CA5 isolated from L cells and endothelial cells; thus, a core protein with a unique primary sequence initiates the synthesis of both GAGs. The expression in L cells of ryudocan12CA5 variants containing a single functional GAG acceptor site demonstrated that each of the three acceptor regions initiates the synthesis of both types of GAGs to a similar extent. Most importantly, in both cell types total HSact generation declined as a function of ryudocan12CA5 overexpression even though HSinact production increased linearly as a function of this variable. This discordant relationship is a general property of the biosynthetic machinery since in both cell types HSact production was reduced to an equal extent on protein cores of either exogenous or endogenous origins. The suppression of HSact generation was also observed with a secreted form of core protein lacking transmembrane and cytoplasmic domains or by a GAG acceptor site mutated form of core protein incapable of augmenting GAG synthesis. These results suggest that elevated intracellular levels of core protein saturate the capacity of a critical component of the HSact biosynthetic machinery. This critical component is not a member of the common set of biosynthetic enzymes involved in the production of HSact and HSinact since no structural changes were observed in either GAG during overexpression of core protein. Based upon the above data, we conclude that increased intracellular levels of ryudocan probably act by saturating the capacity of components which regulate HSact production by coordinating the function of biosynthetic enzymes.