One of thirty murine monoclonal antibodies, raised by immunization with human plasmin-alpha 2-antiplasmin complex, was found to be directed against the high-affinity lysine-binding site in plasminogen. Indeed, this antibody (MA-HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple-loop structures (LBS I) and was displaced by 6-aminohexanoic acid (50% displacement at 25 microM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA-HAL was reduced to 50% with 2.3 microM alpha 2-antiplasmin or 1.3 microM histidine-rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high-affinity lysine-binding site of plasminogen. MA-HAL did not influence the activation of plasminogen by tissue-type plasminogen activator in the absence of CNBr-digested fibrinogen, but abolished the effect of CNBr-digested fibrinogen on the Michaelis constant of the reaction. MA-HAL reduced the reaction rate between plasmin and alpha 2-antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA-HAL specifically binds to and masks the high-affinity lysine-binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin-stimulated activation of plasminogen and of the inhibition of generated plasmin.