Tissue factor (TF) is believed to play an important role in coagulation, inflammation, angiogenesis and wound healing as well as in tumor growth and metastasis. To facilitate in vivo studies in experimental murine models, we have produced recombinant murine factor VII (FVII) and the ectodomain of murine TF, TF(1-223). Murine FVII was activated to FVIIa with human factor Xa and upon reaction with FFR-chloromethyl ketone converted into an active site-blocked TF antagonist, FFR-FVIIa. The activity of murine FVIIa was characterized in factor X activation assays as well as in clot assays with murine and human thromboplastin in murine and human plasma. In these assays murine FVIIa exhibited a specific activity equivalent to or higher than human FVIIa. Further analysis showed that murine FVIIa binds with high affinity to both murine and human TF, whereas the association of human FVIIa to murine TF is about three orders of magnitude weaker than the association to human TF. This difference was further emphasized by the effect of murine-and human FFR-FVIIa on bleeding in an in vivo mouse model. Intra-peritoneal administration of 1 mg/kg murine FFR-FVIIa significantly prolonged the tail-bleeding time, whereas no effect on bleeding was observed with a 25-times higher dose of the human FFR-FVIIa. Together, these data confirms the notion of poor species compatibility between human FVII and murine TF and emphasizes the requirement for autologous FVIIa in studies on the role of the TF in experimental in vivo pharmacology.