The aim of this work was to clarify the role of urokinase-type plasminogen activator (uPA) on the profibrinolytic activity of heparin, chemically modified heparins [partially: N-desulfated (N-des), N-desulfated N-acetylated (N-des N-ac), O-desulfated (O-des), O/N-desulfated N-acetylated (O/N-des N-ac)] and heparan sulfate. Binding competition assays of plasminogen and uPA to heparin-sepharose demonstrated that heparin bound to both enzymes. Moreover, in the presence of increasing amounts of heparin, plasminogen activation mediated by uPA occurred as a bell-shaped curve, suggesting the formation of a ternary complex. In contrast, all chemically-modified heparins lacked this cofactor activity, although N-des and heparan sulfate partially retained the uPA binding capacity, and O-des partially bound to both plasminogen and uPA. Plasmatic euglobulins from mice treated with heparin, as well as with modified heparins with uPA binding capacity, presented a 2-fold enhancement of 47 kDa lytic band, as assessed by zymographic analysis. Western blotting analysis anti-uPA (47 kDa) showed that the enhanced uPA activity correlated with a true increase in uPA protein levels. These results suggest that the profibrinolytic activity of heparin mediated by uPA could be caused by an increase in uPA protein levels rather than by a cofactor activity mediated by a formation of ternary complexes.