TF (tissue factor) is a transmembrane cofactor that initiates blood coagulation in mammals by binding Factor VIIa to activate Factors X and IX. The cofactor can reside in a cryptic configuration on primary cells and de-encryption may involve a redox change in the C-terminal domain Cys¹⁸⁶–Cys²⁰⁹ disulfide bond. The redox potential of the bond, the spacing of the reduced cysteine thiols and their oxidation by TF activators was investigated to test the involvement of the dithiol/disulfide in TF activation. A standard redox potential of −278 mV was determined for the Cys¹⁸⁶–Cys²⁰⁹ disulfide of recombinant soluble TF. Notably, ablating the N-terminal domain Cys⁴⁹–Cys⁵⁷ disulfide markedly increased the redox potential of the Cys¹⁸⁶–Cys²⁰⁹ bond, suggesting that the N-terminal bond may be involved in the regulation of redox activity at the C-terminal bond. Using As(III) and dibromobimane as molecular rulers for closely spaced sulfur atoms, the reduced Cys¹⁸⁶ and Cys²⁰⁹ sulfurs were found to be within 3–6 Å (1 Å=0.1 nm) of each other, which is close enough to reform the disulfide bond. HgCl₂ is a very efficient activator of cellular TF and activating concentrations of HgCl₂-mediated oxidation of the reduced Cys¹⁸⁶ and Cys²⁰⁹ thiols of soluble TF. Moreover, PAO (phenylarsonous acid), which cross-links two cysteine thiols that are in close proximity, and MMTS (methyl methanethiolsulfonate), at concentrations where it oxidizes closely spaced cysteine residues to a cystine residue, were efficient activators of cellular TF. These findings further support a role for Cys¹⁸⁶ and Cys²⁰⁹ in TF activation.