Although vinexin was originally identified as a protein binding to the proline-rich hinge region of vinculin, the functions and biochemical properties of the vinexin-vinculin interaction are not known. Here, we determined the affinity of the vinexin-vinculin interaction using surface plasmon resonance measurements and found that vinexin beta interacts with the C-terminal half of vinculin, which mimics an activated "open" form, with a threefold higher affinity than with the full-length "closed" vinculin. Coimmunoprecipitation experiments showed that cell adhesion on fibronectin enhances the vinexin vinculin interaction. We also show that the interaction with vinculin is necessary for the efficient localization of vinexin alpha and beta at focal adhesions. These observations suggest a model that "activated" vinculin localized at focal adhesions recruits vinexins to focal adhesions. (c) 2005 Elsevier Inc. All rights reserved.