We have constructed and characterized a chimeric anticoagulant and fibrinolytic protein by inserting the portion of thrombomodulin (TM) carrying protein C (PC) activating cofactor activity into tissue-type plasminogen activator (t-PA). This was done by replacing the epidermal growth factor (EGF)-like domain of t-PA with the six EGF-like repeats from TM. The 105 kDa chimeric molecule was secreted from transfected COS-7 cells as a single-chain molecule that was recognised by both anti-TM and anti-t-PA antisera. It was shown to have similar PC activating cofactor activity to other soluble derivatives of TM (Km for Protein C of 2.2±0.4 μM), as well as amidolytic and plasminogen activating activity. However, the chimeric protein is not converted to a two chain molecule and is stimulated less by fibrin, due to a lower affinity for lysine. The demonstration that TM activity can be incorporated into a fibrinolytic enzyme is a step towards enhanced fibrinolysis in patients with thrombosis.