Thiol-disulfide isomerization in thrombospondin may affect the function of this adhesive protein. Two assays were developed to analyze the determinants of thiol-disulfide exchange and to correlate this exchange with thrombospondin conformation. (1) A competitive immunoassay for the EDTA-conformation of thrombospondin was developed with monoclonal antibody D4.6. (2) The free thiol(s) in thrombospondin was labeled with [³H]N-ethylmaleimide (NEM) under various conditions (the presence or absence of calcium, temperature, and pH), and thrombin digests of the labeled protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Consistent with previous reports, thrombin digest fragments of 150, 120, 20, and 14 kD were observed, each with radioactivity under some condition, plus a 25-kD peptide that was not labeled. Sequence data for these fragments and comparisons of SDS-PAGE analyses under reducing and nonreducing conditions indicated that Cys⁹⁷⁴ was the free thiol. The appearance of thiol label in the 120-kD fragment was previously shown to be a consequence of thiol-disulfide exchange (J Biol Chem 265:17859,1990) and label was recovered in this peptide only under conditions (absence of calcium, 37°C and pH 8.4) that led to the appearance of the EDTA-conformation of thrombospondin. Additional evidence for the correlation of EDTA-conformation and thiol-disulfide exchange was the enhanced conversion of thrombospondin to its EDTA-conformation in the presence of protein disulfide isomerase and the inability of thrombospondin pretreated with NEM to attain the EDTA-conformation. Flow cytometry with antibody D4.6 revealed platelet-associated thrombospondin in the EDTA-conformation in the presence of calcium, suggesting that the EDTA-conformation is a physiological conformation that does not necessarily require EDTA.