Summary Defibrotide is a polyanionic deoxyribonucleotide extract that has demonstrated beneficial actions in thrombotic disease. This agent is free from anticoagulant activity but profibrinolytic effects have been claimed following both oral and intravaenous administration. The aim of the present study was to characterize further the profibrinolytic action of defibrotide in the rat and investigate the mechanism underlying this response. Following administration of defibrotide (25–200 mg/kg) to laboratory rats, a market, dose-dependent shortening of the plasma euglobulin clot lysis time was observed, accompanied by significant decreases in plasma plasminogen activator inhibitor (PAI) activity at 30 min post dosing but no change in tissue-type plasminogen activator (t-PA) activity. Inhibition of plasma eicosanoid generation in vivo using the cyclo-oxygenase inhibitor indomethacin (25 mg/kg i.p. 45 min prior to experiment) had no effect on this defibrotide-induced profibrinolytic state. In vitro studies using cultured human umbilical vein endothelial cells failed to demonstrate any direct effect of defibrotide on either the secretion or activity of t-PA or PAI. Pentosan sulphate, another polyanionic agent with claimed utility for the treatment of thrombotic disorders, also induced a marked profibrinolytic response evidenced by reduced euglobulin clot lysis time and decreased plasma PAI activity. We conclude that polyanionic agents such as defibrotide and pentosan sulphate increase plasma fibrinolytic activity through substantial reductions in plasma PAI activity. This effect is achieved by an undefined mechanism of action which is not related to either induction of eicosanoid synthesis or by a direct effect on the secretion of t-PA or PAI at the level of the vascular enthothelial cell.