When blood is clotted, alpha(2)-plasmin inhibitor (alpha(2)PI) is cross-linked to fibrin by activated fibrin-stabilizing factor (activated coagulation Factor XIII, plasma transglutaminase). The amount of cross-linked alpha(2)-PI is proportional to the amount of alpha(2)PI present at the time of clotting. Plasma from a patient with congenital deficiency of alpha(2)PI was supplemented with various amounts of purified alpha(2)PI. Clots were prepared from these plasmas and were suspended in plasma containing a normal concentration of alpha(2)PI, and spontaneous clot lysis was observed. When the clot was formed in the presence of calcium ions and thereby allowing cross-linking to occur, the rate and extent of fibrinolysis were found to be inversely proportional to the concentrations of alpha(2)PI present in the clot at the time of clotting. When the clot was formed in the absence of calcium ions so that no cross-linking occurred, the clot underwent fibrinolysis at similar rates, regardless of the concentrations of alpha(2)PI in the clot. When the clot formed in the presence of calcium ions was squeezed and washed to remove unbound proteins before being suspended in plasma, the extent of fibrinolysis was also inversely proportional to the amount of alpha(2)PI cross-linked to fibrin. Similar results were obtained when the clot was suspended in buffered saline instead of plasma. These observations suggest that spontaneous fibrinolysis is mainly carried out by plasminogen/plasminogen activator bound to fibrin, and this fibrinolysis caused by fibrin-associated activation of plasminogen was mainly inhibited by alpha(2)PI cross-linked to fibrin. To further support this concept, alpha(2)PI treated with activated fibrin-stabilizing factor and that had lost most of its cross-linking capacity was used in similar experiments. This modified alpha(2)PI had the same inhibitory activity on plasmin as the native inhibitor, but gave significantly less inhibition of fibrinolysis in every experiment, particularly when the clot was compacted by platelet-mediated clot retraction or by squeezing. Thus, it was concluded that alpha(2)PI cross-linked to fibrin plays a significant role in inhibition of physiologically occurring fibrinolysis. It is further suggested that the absence of cross-linked alpha(2)PI contributes to accelerated fibrinolysis and hemorrhagic tendency in patients with congenital deficiency of fibrin-stabilizing factor.