Integrins are adhesion molecules that convey signals both to and from the cytoplasm across the plasma membrane. In resting cells, integrins in a low affinity state can be activated by'inside-out signaling', in which signals affecting integrin heterodimer cytoplasmic domains cause a conformational change in the integrin ligand-binding headpiece connected to the membrane by two long,∼ 16 nm stalks. Here we demonstrate a mechanism for conveying a conformational change over the long distance from the plasma membrane to the headpiece. We prepared soluble, α5β1 integrin heterodimer extracellular fragments in which interactions between α-and β-subunit cytoplasmic domains were replaced with an artificial clasp. Release of this C-terminal clasp by specific protease cleavage resulted in an∼ 14 nm separation of the stalks coupled to increased binding to fibronectin. This activation did not require any associated molecules or clustering and was observed with physiological concentrations of divalent cations. These findings suggest that the overall mechanism for integrin inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains.