Fibronectin immobilized onto polystyrene surface was found to bind plasminogen and tissue-type plasminogen activator (t-PA) but only slightly the urokinase type as determined using mono- and polyclonal antibodies against the activators. Of the defined fibronectin fragments tested, the Mr 120,000-140,000 fragment was found to bind both plasminogen and t-PA. Proteolytically modified plasminogen (Lys-plasminogen) bound considerably better than the native form (Glu-plasminogen). Experiments with 125I-plasminogen yielded Kd = 9.1 X 10(-8) M for the binding to immobilized fibronectin. The partially or completely inactive single-chain form of t-PA (pro-t-PA) bound considerably better than the activated two-chain form. Lysine at greater than 3 mM inhibited the binding of plasminogen. The interaction was independent of calcium ions. CaCl2 (greater than 0.5 mM) and NaCl (greater than 0.2 M) inhibited the binding of pro-t-PA and of t-PA. Fibronectin-bound t-PA retained its ability to activate plasminogen. The observed interactions may operate in directional proteolysis localizing plasminogen and plasminogen activator to degrade fibronectin-containing extracellular matrix including fibrin clots.