Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme that after activation down-regulates fibrinolysis. Platelets are known to contain antifibrinolytic factors that are secreted during platelet activation. Therefore, the presence of TAFI in platelets was analyzed. TAFI was identified in platelets in a concentration of about 50 ng/1 × 10⁹ platelets and was secreted on platelet activation. Thrombin-mediated activation of platelet-derived TAFI resembled that of plasma-derived TAFI with respect to stimulation by thrombomodulin and spontaneous loss of activity at 37°C. The different glycosylation of platelet-derived TAFI compared with plasma-derived TAFI suggests that platelet-derived TAFI is synthesized in the megakaryocyte. This suggestion was substantiated by the detection of mRNA in the megakaryocytic cell lines DAMI and CHRF, representing the intermediate and late stages of megakaryocyte development. These results establish the presence of TAFI in platelets and suggest a role for platelet-derived TAFI in the protection of the clot against fibrinolysis.