Ancrod, a thrombin-like enzyme, has been used as defibrinogenating agent in prevention of venous thrombosis. This study showed ancrod in citrated plasma elevated 6-keto PGF1 alpha production of human umbilical vein endothelial cells; this increment of 6-keto PGF1 alpha was completely inhibited by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The enhanced prostacyclin production, but not basal level of prostacyclin, was inhibited by actinomycin D and cycloheximide. In washed aspirin-pretreated cells, ancrod-formed fibrin induced restoration of prostacyclin production by a cycloheximide- and actinomycin D-sensitive process. Ancrod-formed fibrin stimulated synthesis of cyclooxygenase as probed by Western blotting and this enhancement was blocked by actinomycin D and cycloheximide. In conclusion, we first report that ancrod-formed fibrin stimulates prostacyclin production of human endothelial cells and this event is dependent on de novo synthesis of cyclooxygenase.