Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound ␣ 5 and ␤ 1 integrin subunits but not ␣ v or ␤ 3 . C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B Ͼ T Ͼ C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of ␣ 5 ␤ 1 integrin bound to Fn, and differentiation was inhibited by anti-␣ 5 , but not anti-␣ v , antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. INTRODUCTIONThe adhesion of cells to their substrate through an extracellular matrix provides signals that influence their ability to survive, proliferate, and express specific developmental phenotypes (Menko and Boettiger, 1987;Werb et al., 1989;Adams and Watt, 1990;Streuli et al., 1991;Zhu et al., 1996;Chen et al., 1997). One of the early examples was the development of in vitro culture conditions that permitted the differentiation of avian myogenic cells into contracting myotubes (Hauschka and Konigsberg, 1966;Bischoff and Holtzer, 1968). The critical element for this system was the precoating of tissue culture surfaces with rat tail collagen. This general principle of providing an appropriate substrate to permit the expression of developmental phenotypes has been applied to a wide variety of cells. These include systems that allow the maintenance of neurons and outgrowth of growth cones (Westerfield, 1987) and the recapitulation of the stages of mammary gland development and involution (Li et al., 1987;Barcellos-Hoff et al., 1989). These findings indicate that critical elements of the message directing the expression of a differentiated phenotype are encoded in the extracellular matrix.Cells interact with extracellular matrices primarily through integrins, a widely expressed family of cell surface receptors (Hynes, 1987), and integrin binding to its extracellular ligand is responsible for the downstream effects of the matrix on cell function. For example, in the muscle differentiation syste...