Integrins play a key role in mechanobiology due to their linkages with the extracellular matrix (ECM) and cytoskeleton making the mechanical characteristics and properties of integrin behavior important for understanding the molecular nature of how cells interact with their mechanical environment. Through the process of mechanotransduction, mechanical forces act on vascular smooth muscle cells (VSMC) at the level of assembled focal adhesions that are associated with downstream signaling events that can be of particular significance in cardiovascular disease. The goal of this study was to characterize the mechanical properties of the initial fibronectin-₅₁ interaction in VSMC using Atomic Force Microscopy (AFM). It is postulated that these initial binding events are important to the subsequent process of focal adhesion assembly that ultimately provides the cell with the ability to sense and respond to mechanical forces. Specifically, AFM was used to measure the unbinding force and binding activity between integrins and fibronectin (FN) on the surface of VSMC isolated from rat cremaster arterioles (70-100 µm diameter). Function blocking antibodies against ₅ and ₁ integrins as well as RGD-containing peptides selectively blocked FN-VSMC adhesions. Function blocking antibodies for ₄ and ₃ integrin had no significant blocking effect, indicating that FN primarily bound to ₅₁ on the surface of VSMC. A characteristic unbinding force of 39±8 pN was observed, and interpreted to represent the single bond strength between FN and ₅₁ integrin. The ability of FN adhere to the surface of VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet derived growth factor (PDGF-BB), while lysophosphatidic acid (LPA) increased FN binding. However, these treatments induced no significant changes in the resolved unbinding force. When an FN-coated bead was brought into contact with the VSMC surface, the force required to dislodge the bead from the cell increased with increasing time of contact with the cell. This indicated a time-dependent and progressive increase in adhesion between the FN coated bead and the cell, suggesting an increased number of adhesion and/or altered binding affinity characteristics. LPA enhanced the time-dependent binding and adhesion between the FN coated bead and the cell, while PDGF reduced it suggesting that these factors also affect the multi-molecular process of focal contact assembly. Thus, AFM is a useful and powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of ₅₁ and focal contact assembly can be rapidly regulated.