First generation thrombolytics (streptokinase and urokinase) had no fibrin binding capabilities and caused systemic plasminogen activation with concomitant destruction of haemostatic proteins. A primary driving force behind the development of the second generation plasminogen activator tissue plasminogen activator (tPA or alteplase) was its ability to bind to fibrin and target thrombolysis. Although in vitro assays highlighted advantages of fibrin binding, clinical trials were disappointing, showing only small benefits in mortality with tPA versus streptokinase, but also with some increase in haemorrhagic stroke. Third generation thrombolytic agents (reteplase, tenecteplase and pamiteplase) are variants of tPA engineered to have improved structure/function, such as longer half life and resistance to inhibitors. However, clear therapeutic advantages of third generation thrombolytics in clinical trials have also been difficult to demonstrate. Although fibrin binding is critical in regulating the activity of tPA, it is not clear how important it is for thrombolytic treatment. Advances are needed in our understanding of the relationship between structure/binding and activity of PAs in vivo under normal conditions and when administered in pharmacological doses. Clearly the impact of fibrin structure and the other components in fibrin clots must also be considered. Ultimately these studies may lead to better engineered therapeutics or optimised mixtures of molecules. With a more detailed understanding of the regulation of plasminogen activation and fibrinolysis it might be possible to tailor thrombolytic therapy to different situations such as myocardial or cerebrovascular treatment or to the patient's age and sex and other characteristics.