The carboxypeptidase, TAFIa or CPU, is known to prolong plasma clot lysis by tissue plasminogen activator (tPA) and to have a role in thrombus stability in vivo. This current study examined lysis by urokinase (uPA) and single chain urokinase (scuPA) in addition to tPA. Further, we investigated the role of TAFIa in a model thrombus system, in which thrombi are formed under conditions of flow. We show that human thrombi, formed in vivo, and model thrombi both contain TAFI. No effect of thrombus TAFIa was observed in thrombus lysis assays, except when thrombi were bathed in plasma, in which case addition of potato tuber carboxypeptidase inhibitor (CPI) resulted in doubling of the rate of lysis. TAFIa inhibited lysis of model thrombi and plasma clots by uPA, scuPA in addition to lysis by tPA. The effect of TAFIa was more evident at high concentrations of plasminogen activator such as those used in thrombolytic therapy. Addition of plasminogen increased lysis and, in its presence, the enhancement by CPI was smaller. Thus the action of TAFIa could be partially overcome by plasminogen, whether lysis was by tPA, uPA or scuPA. This is consistent with TAFIa exerting its effect primarily through modifying the binding of plasminogen to fibrin and to a lesser extent through modification of the binding of tPA to fibrin.