Retroviral vector-mediated expression of plasminogen activators (PAs) from endothelial cells (ECs) has been proposed as a potential therapeutic approach for intravascular thrombosis. To define the potential for gene transfer to increase fibrinolytic activity in a primate system, baboon ECs were transduced with retroviral vectors expressing wild-type and glycosylphosphatidylinositol-anchored urokinase, as well as wild-type and serpin-resistant tissue PA (t-PA). Expression of either t-PA or urokinase was increased by one log over baseline levels. There was no specific effect of either t-PA or urokinase overexpression on endogenous t-PA, urokinase, or PA inhibitor 1 (PAI-1) expression. Recombinant urokinase could be anchored to the cell surface at a level eight-fold above that of receptor-bound urokinase. The majority of secreted urokinase accumulated in conditioned medium as a free proenzyme, whereas both wild-type and serpin-resistant t-PA accumulated almost exclusively in complexes with PAI-1. In most but not all of the assays, the urokinase vectors conferred PA activity above that of the t-PA vectors. These data show that PA synthesis and activity are specifically increased subsequent to retroviral vector-mediated gene transfer in primate ECs. However, definition of an optimal PA vector will require in vivo experimentation.