The kinetics of inhibition of the amidolytic activity of plasmin on D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) by fibrinogen and fibrin were determined. Reciprocal (1/v versus 1/[S]) plots of plasmin inhibition by 0.50 microM-fibrinogen showed a non-linear downward curve. The Hill coefficient (h) was 0.68, suggesting negative co-operativity. By contrast, fibrin produced a simple competitive inhibition of plasmin (Ki = 12 micrograms/ml). Addition of 0.1 mM-6-aminohexanoic acid shifted the non-linear curve obtained in the presence of fibrinogen to a straight line as for controls, indicating that 6-aminohexanoic acid abolishes the fibrinogen-induced inhibition. Transient exposure of the enzyme to pH 1.0 abrogates the ability of fibrinogen to inhibit plasmin activity. Acidification had no effect on the Vmax but increased the Km of plasmin. The present evidence for modulation of plasmin reveals a novel mechanism for control of fibrinolysis by fibrinogen, a component of the coagulation system and the precursor of the physiological substrate of plasmin.