The role of vitronectin (Vn) in thrombosis is currently controversial; both inhibitory and supportive roles have been reported. To monitor directly the function of Vn in thrombotic events at the site of vascular injury, we studied Vn-deficient (Vn-/-) and wild-type (WT) control mice with two real-time intravital microscopy thrombosis models. In the mesenteric arteriole model, vessel injury was induced by ferric chloride. We observed unstable thrombi and a significantly greater number of emboli in Vn-/- mice. Vessel occlusion was also delayed and frequent vessel re-opening occurred. In the cremaster muscle arteriole model, vessel injury was induced by a nitrogen dye laser. We observed significantly fewer platelets, lower fibrin content, and unstable fibrin within the thrombi of Vn-/- mice. To define further the role of Vn in thrombus growth, we studied platelet aggregation in vitro. Consistent with our in vivo data, the second wave of thrombin-induced aggregation of gel-filtered platelets was abolished at a low concentration of thrombin in Vn-/- platelets. Interestingly, adenosine diphosphate (ADP)-induced platelet aggregation was significantly increased in Vn-/- platelet-rich plasma (PRP) and this effect was attenuated by adding purified plasma Vn. We also observed increased platelet aggregation induced by shear stress in Vn-/- whole blood. These data demonstrate that Vn is a thrombus stabilizer. However, in contrast to released platelet granule Vn which enhances platelet aggregation, plasma Vn inhibits platelet aggregation.