Bone morphogenetic proteins (BMPs) are involved in multiple aspects of limb development including regulation of cartilage differentiation. Several BMPs bind strongly to heparin, and heparan sulfate proteoglycans (HSPGs) at the cell surface or in the extracellular matrix have recently been implicated as modulators of BMP signaling in some developing systems. Here we have explored the role of HSPGs in regulating BMP activity during limb chondrogenesis by evaluating the effects of exogenous heparan sulfate (HS), heparitinase treatment, and overexpression of the HSPG syndecan-3 on the ability of BMP2 to modulate the chondrogenic differentiation of limb mesenchymal cells in micromass culture. Exogenous HS dramatically enhances the ability of BMP2 to stimulate chondrogenesis and cartilage specific gene expression, and reduces the concentration of BMP2 needed to stimulate chondrogenesis. Furthermore, HS stimulates BMP2-mediated phosphorylation of Smad1, Smad5, and Smad8, transcriptional mediators of BMP2 signaling, indicating that HS enhances the interaction of BMP2 with its receptors. Pretreatment of micromass cultures with heparitinase to degrade endogenous HSPGs also enhances the chondrogenic activity of BMP2, and reduces the concentration of BMP2 needed to promote chondrogenesis. Taken together these results indicate that exogenous HS or heparitinase enhance the chondrogenic activity of BMP2 by interfering with its interaction with endogenous HSPGs that would normally restrict its interaction with its receptors. Consistent with the possibility that HSPGs are negative modulators of BMP signaling during chondrogenesis, we have found that overexpression of syndecan-3, which is one of the major HSPGs normally expressed during chondrogenesis, greatly impairs the ability of BMP2 to promote cartilage differentiation. Furthermore, retroviral overexpression of syndecan-3 inhibits BMP2-mediated Smad phosphorylation in the regions of the cultures in which chondrogenesis is inhibited and in which ectopic syndecan-3 protein is highly expressed. These results indicate that syndecan-3 interferes with the interaction of BMP2 with its receptors, and that this interference results in an inhibition of chondrogenesis. Taken together these results indicate that HSPGs including syndecan-3 normally modulate the strength of BMP signaling during limb cartilage differentiation by limiting the effective concentration of BMP available for signaling.