Fibrinolytic activity of normal plasma and blood has been measured by 125l-fibrin solid phase assay. Activity of plasma is not affected by removal of plasminogenplasmin by affinity chromatography. Activities of euglobulin and pseudoglobulin fractions are approximately equal. epsilon-aminocaproic acid (EACA) (10 mM), tranexamic acid (10 mM), diisopropylfluorophosphate (DFP, 50 mM), and soybean and lima bean trypsin inhibitors (100 mug/ml) do not inhibit plasma activity at concentrations that inhibit pure plasmin and urokinase-activated plasma. Activity is not affected by glass contact and is not inhibited by inhibitors of contact or enzymatic activation of Hageman factor (hexadimethrine bromide, 100 mug/ml; cytochrome C, 250 mug/ml; spermidine, 2 mM; phenylmethylsulfonylfluoride, 1 mM). It is inhibited partially (30%-40%) by heating (56 degrees C, 30 min) and by zymosan (2.5 mg/ml; 40%-90% inhibition), and is increased by hydrazine (20 mM), salicylaldoxime (20 mM), DFP (50 mM), and tosyl-L-arginine methyl ester (TAMe, 10 mM)-the latter two at concentrations known to inhibit Cls of the classic, and factor D of the alternate complement pathways. Increase fibrinolytic activity with TAMe is associated with reciprocal decrease in classic and alternate complement pathway activity. It is concluded that normal plasma fibrinolytic activity is relatively independent of plasmin as the ultimate fibrinolytic enzyme, that Hageman factor-dependent pathways are of minor importance, and that significant heat-stable and heat-labile nonplasmin fibrinolytic activities are operative. These may include proteinases involved in complement activation, and in common control of classic and alternate complement pathways, as well as other nonplasmin proteinases.