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Latent transforming growth factor-β-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGFβ in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-β (TGFβ) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGFβ into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full …