Plasmin(ogen) kringles 1 and 4 are involved in anchorage of plasmin(ogen) to fibrin and cells, an essential step in fibrinolysis and pericellular proteolysis. Their contribution to these processes was investigated by selective neutralization of their lysine-binding function. Blocking the kringle 1 lysine-binding site with monoclonal antibody 34D3 fully abolished binding and activation of Glu-plasminogen and prevented both fibrinolysis and plasmin-induced cell detachment-induced apoptosis. In contrast, blocking the kringle 4 lysine-binding site with monoclonal antibody A10.2 did not impair its activation although it partially inhibited plasmin(ogen) binding, fibrinolysis and cell detachment. This remarkable, biologically relevant, distinctive response was not observed for plasmin or Lys-plasminogen; each antibody inhibited their binding and activation of Lys-plasminogen to a limited extent, and full inhibition of fibrinolysis required simultaneous neutralization of both kringles. Thus, in Lys-plasminogen and plasmin, kringles 1 and 4 act as independent and complementary domains, both able to support binding and activation. We conclude that Glu-/Lys-plasminogen and plasmin conformations are associated with transitions in the lysine-binding function of kringles 1 and 4 that modulate fibrinolysis and pericellular proteolysis and may be of biological relevance during athero-thrombosis and inflammatory states. These findings constitute the first biological link between plasmin(ogen) transitions and functions.