The state of the five half-cystine residues in human tissue factor (TF) has been characterized. the results indicate that the four half-cystines in the extracellular domain of TF form two disulfide bonds and the half-cystine in the cytoplasmic region is acylated by palmitic acid and stearic acid. the extracellular disulfide cross-links, Cys49-Cys57and Cys186-Cys209, were deduced from the analysis of tryptic peptides. Acylation of the cytoplasmic half-cystine was demonstrated by purifying and characterizing fibroblast TF from cells labeled with [3H] palmitic acid. Radiolabeled fibroblast TF was observed by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. the tritiated material covalently bound to the protein was identified as [3H] palmitate and [3H] stearate by reverse-phase high-pressure liquid chromatography. Deacylation of TF with hydroxylamine resulted in the spontaneous generation of di-sulfide-linked TF dimers. This result suggests that the disulfide-linked TF dimer, a minor component of most TF preparations, and the recently described heterodimeric form of TF are artifacts produced by deacylation of Cys245and subsequent interchain disulfide bond formation.