Integrins are the major family of receptors involved in the adhesive interactions of cells with extracellular matrix macromolecules. Although it is known that integrins can exist in active or inactive states, the molecular mechanisms by which integrin activity is modulated are poorly understood. A novel anti‐integrin monoclonal antibody, 12G10, that enhances α5β1‐fibronectin interactions has been identified. 12G10 binds to the β1 subunit and appears to recognise a region of the subunit that contains the epitopes of several previously described activating or inhibitory monoclonal antibodies. However, unlike other activating anti‐β1 antibodies, the binding of 12G10 to α5β1 is increased in the presence of ligands (fibronectin fragment or RGD peptide). This is the first report for the β1 integrin family of an antibody that recognises a ligand‐induced binding site, and further emphasises the functional importance of a specific region of the β1 subunit in regulating integrin‐ligand interactions.