Microvessels are composed of endothelial cells that cover the luminal surface and pericytes that wrap around and along endothelial cells. In the present study, we investigated the regulation of fibrinolysis in cultured human brain microvascular pericytes and endothelial cells after exposure to thrombin. It was found that the regulation in pericytes is different from that in endothelial cells. Specifically, thrombin increases the secretion of fibrinolytic proteins (tissue- and urokinase-type plasminogen activators and plasminogen activator inhibitor-1), resulting in an enhancement of plasminogen activator activity in endothelial cells, whereas the proteinase increases the secretion of only plasminogen activator inhibitor-1 by activation of proteinase-activated receptor-1 and induces suppression of plasminogen activator activity in pericytes. The present data suggest that thrombin regulation of fibrinolytic activity in endothelial cells and pericytes may be involved in the removal of intravascular thrombi and the maintenance of hemostasis, respectively, in human brain microvessels.