Transforming growth factor‐βs (TGF‐β) are secreted as latent complexes consisting of the TGF‐β dimer, the TGF‐β propeptide dimer, and the latent TGF‐β binding protein (LTBP). Although the bonds between TGF‐β and its propeptide are cleaved intracellulary, the propeptide associates with TGF‐β by electrostatic interactions, thereby conferring latency to the complex. We reported that a specific sequence of LTBP‐1 is required for latent TGF‐β activation by the integrin αvβ6. Here we describe a 24 amino acid sequence from the hinge domain required for activation. The LTBP‐1 polypeptide rL1N, which includes the hinge, associates with fibronectin in binding assays. We present evidence that fibronectin null cells minimally activate latent TGF‐β and poorly incorporate the active hinge sequence into their matrix. In addition, cells missing the fibronectin receptor α5β1 exhibit defective activation of latent TGF‐β by αvβ6 and decreased matrix incorporation. The results indicate specificity for integrin‐mediated latent TGF‐β activation that include unique sequences in LTBP‐1 and an appropriate matrix molecule. Fontana, L., Chen, Y., Prijatelj, P., Sakai, T., Fässler, R., Sakai, L. Y., Rifkin, D. B. Fibronectin is required for integrin αvβ6‐mediated activation of latent TGF‐β complexes containing LTBP‐1. FASEB J. 19, 1798–1808 (2005)