Occlusive thrombosis depends on the net balance between platelets, coagulation, and fibrinolytic factors. Epidemiologic information suggests that plasminogen activator inhibitor-1 (PAI-1), a central regulator of the fibrinolytic system, plays an important role in determining the overall risk for clinically significant vascular thrombosis. Vitronectin (VN), an abundant plasma and matrix glycoprotein, binds PAI-1 and stabilizes its active conformation. This study assessed the role of PAI-1 and VN expression in the formation of occlusive vascular thrombosis following arterial or venous injury. The common carotid arteries of 17 wild-type (WT) mice and 8 mice deficient in PAI-1 were injured photochemically while blood flow was continuously monitored. WT mice developed occlusive thrombi at 52.0 ± 3.8 minutes (mean ± SEM) following injury; mice deficient in PAI-1 developed occlusive thrombosis at 127 ± 15 minutes (P < .0001). Mice deficient in VN (n = 12) developed vascular occlusion 77 ± 11 minutes after injury, intermediate between the values observed for WT mice (P < .03) and mice deficient in PAI-1 (P < .01). PAI-1 and VN also affected the time to occlusion after injury to the jugular vein. Three WT mice developed occlusive venous thrombosis an average of 39.7 ± 1 minutes following the onset of injury, whereas the jugular veins of 4 mice deficient in PAI-1 and 4 deficient in VN occluded 56.7 ± 5 and 58.7 ± 2 minutes, respectively, following injury (P < .04 andP < .01 compared to WT mice). These results suggest that endogenous fibrinolysis and its regulation by PAI-1 and VN have important roles in the development of occlusive vascular thrombosis after vascular injury.