Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B that can down-regulate fibrinolysis. TAFIa is a labile enzyme that can be inactivated by conformational instability or proteolysis. TAFI is approximately 40% identical to pancreatic carboxypeptidase B (CPB). In contrast to TAFIa, pancreatic CPB is a stable protease. We hypothesized that regions or residues that are not conserved in TAFIa compared with pancreatic CPB play a role in the conformational instability of TAFIa and that replacement of these non-conserved residues with residues of pancreatic CPB would lead to a TAFIa molecule with an increased stability. Therefore, we have expressed, purified, and characterized two TAFI-CPB chimeras: TAFI-CPB-(293-333) and TAFI-CPB-(293-401). TAFI-CPB-(293-333) could be activated by thrombin-thrombomodulin, but not as efficiently as wild-type TAFI. After activation, this mutant was unstable and was hardly able to prolong clot lysis of TAFI-deficient plasma. Binding of TAFI-CPB-(293-333) to both plasminogen and fibrinogen was normal compared with wild-type TAFI. TAFI-CPB-(293-401) could be activated by thrombin-thrombomodulin, although at a lower rate compared with wild-type TAFI. The activated mutant displayed a markedly prolonged half-life of 1.5 h. Plasmin could both activate and inactivate this chimera. Interestingly, this chimera did not bind to plasminogen or fibrinogen. TAFI-CPB-(293-401) could prolong the clot lysis time in TAFI-deficient plasma, although not as efficiently as wild-type TAFI. In conclusion, by replacing a region in TAFI with the corresponding region in pancreatic CPB, we were able to generate a TAFIa form with a highly stable activity.