Tissue Factor (TF), a small transmembrane glycoprotein, is the cellular receptor for the zymogen Factor VII and the serine protease Factor VIIa (VIIa). TF provides cofactor function for VIIa in the catalytically active (TF: VIIa) binary complex. To explore the structural loci of TF that are responsible for binding of VII and VIIa, monoclonal antibodies (MAbs) and sequence-specific polyclonal antibodies to the native TF protein were analysed for inhibition of VII binding. Two independent epitopes of MAbs were localized by reciprocal competition and by binding of the MAbs to different proteolytic fragments of TF. The epitopes were also characterized in part by progressive C-terminal deletional mutation of the TF protein. Reactivity of the anti-(locus II) MAb TF9-6G4 is consistent with epitope localization in residues Thr40-Val83, encoded by exon 3. In contrast, the anti-(locus I) MAb TF9-5G9 was reactive with fragments encompassing exon 4 (Thr106-Lys165). Antibodies to linear sequences encoded by the same two exons also inhibited VII binding. These data suggest a minimum requirement for two of the four exon-encoded regions of TF for the functional integrity of this receptor cofactor with respect to ligand recognition and high-affinity binding.