Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin alpha IIb beta 3 Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human alpha IIb beta 3) to a constitutively active form of alpha IIb beta 3 (alpha 6B beta 3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1 Overexpression of an active form of vinculin without intra molecular interactions, but not the full length one, induced PAC-1 binding to native alpha IIb beta 3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of alpha 6B beta 3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates alpha IIb beta 3 Thus, an active form of vinculin could induce alpha IIb beta 3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling (C) 2010 Elsevier Inc All rights reserved.