The binding of human 125I-Glu-plasminogen to human plasmin-degraded fibrin was studied. Treatment of preformed and polymerized fibrin with 0.01 IU plasmin/ml resulted in an increased binding of 125I-Glu-plasminogen depending upon the length of time of preincubation of fibrin with plasmin. Binding reached a plateau of 30% of total added radioactivity after 60 min. At this time, less than 10% of fibrin had been digested. Polyacrylamide/urea/acetic acid gel electrophoresis revealed that the radioiodinated plasminogen bound to plasmin-degraded fibrin was of the Glu form. Computerized non-linear regression analysis of the binding experiments revealed that limited plasmic degradation of fibrin progressively generates high-affinity binding sites (Kd approximately equal to 0.3 microM) for Glu-plasminogen. At the time of maximal Glu-plasminogen binding approximately 5 high-affinity binding sites per 100 molecules of fibrin had been generated. The low-affinity type of binding sites were also identified. These observations describe a new mechanism which exquisitely modulates the plasmic breakdown of fibrin by a continuous renewal of high-affinity binding sites for Glu-plasminogen on the surface of the fibrin gel during the fibrinolytic process.