Focal adhesions are randomly distributed across the ventral surface or along the edge of epithelial cells. In fibroblasts they orient centripetally and concentrate at a few peripheral sites connecting long F-actin stress fibers, causing a typical elongated, contractile morphology. Extensive remodeling of adhesions in fibroblasts also takes part in fibronectin fibrillogenesis, a process that depends on Rho-mediated contractility and results in the formation of a fibronectin matrix. Our current study shows that all these fibroblast characteristics are controlled by the ability of integrin α5β1 to bind soluble fibronectin molecules in their compact inactive conformation. The hypervariable region of the ligand-binding I-like domain of integrin α5β1 supports binding of soluble fibronectin. This supports the distribution of centripetally orientated focal adhesions in distinct peripheral sites, Rho activation and fibronectin fibrillogenesis through a mechanism that does not depend on Syndecan-4. Integrin αvβ3, even when locked in high affinity conformations for the RGD recognition motif shows no appreciable binding of soluble fibronectin and, consequently, fails to support the typical fibroblast focal adhesion distribution, Rho activity and fibronectin fibrillogenesis in the absence of integrin α5β1. The ability of α5β1 integrin to interact with soluble fibronectin may thus drive the cell-matrix adhesion and cytoskeletal organization required for a contractile, fibroblast-like morphology, perhaps explaining why α5β1 integrin, similarly to fibronectin, is essential for development.