Latent transforming growth factor beta binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-ß in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-ß into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulphate proteoglycans (HSPGs) may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N-terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N-terminus of LTBP1 between residues 345 to 487. Solid phase binding assays suggest that LTBP1 does not bind directly to fibronectin, but that the binding is indirect. Heparin BSA was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-ß in the extracelluar matrix, with increased amounts of soluble latent TGF-ß. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by ß-D-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that HSPGs may play a critical role in regulating TGF-ß availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin.