The oxidation of captopril (CpSH, 1-(D-3-mercapto-2-methylpropanoyl)-1-proline) by glutathione disulfide (GSSG) via thiol/disulfide exchange to form, in the first step, CpSSG and GSH and, in the second step, CpSSCp and GSH, has been studied in aqueous solution by1H nmr. Due to slow rotation around the amide bond(s) of CpSH and CpSSCp and of the captopril part of CpSSG, separate resonances are observed for thecisandtransconformations across these bonds. Conformational equilibrium constants were estimated as a function of pH for CpSH, CpSSCp, and CpSSG from the intensities of resonances for thecisandtransisomers. These equilibrium constants were used in the determination of equilibrium constants for the two steps in the oxidation of CpSH by GSSG. The results suggest that CpSH has a greater tendency to reduce disulfide bonds by thiol/disulfide exchange at physiological pH, and thus form mixed disulfides, than do the thiol groups in amino acids. Also, the conformational equilibrium constants indicate that, at physiological pH, approximately two thirds of the captopril, either free or in a disulfide form, has thetransconformation.