While incorporation of penicillamine residues (Pen; beta,beta-dimethyl cysteine) into a peptide can cause dramatic changes in biological activity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the beta-methyls as a synthetic device to control the production of disulfide isomers. That is, oxidation of a peptide containing an equal number of Cys and Pen residues should predominantly form products which contain mixed Cys-Pen disulfides. Endothelin (ET) is a 21 amino acid peptide which contains Cys at positions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been reported to produce a 3:1 ratio of the natural 1-15, 3-11 to the unnatural 1-11, 3-15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products containing Cys-Pen disulfides. Random oxidation (air, aqueous NH4OH) of the tetrathiols of [Pen1,11, Nle7]-ET-1 or [Pen3,15, Nle7]-ET-1 produced the correct 1-15, 3-11 isomer in > 12:1 and > 22:1 ratios, respectively. Oxidation of the tetrathiol of [Pen1,15, Nle7]-ET-1 favored the unnatural 1-11, 3-15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product as a result of the driving force for penicillamine mixed disulfide formation. Disulfide isomers were identified using ion-spray mass spectrometry in conjunction with enzymatic and acid hydrolysis. [Pen1,11, Nle7]-ET-1 was a partial agonist at the ETA receptor (EC50 = 7.5 nM in rabbit carotid artery rings; Kd = 4.5 nM in rat A10 cell membranes) while [Pen3,15, Nle7]-ET-1 (EC50 = 0.9 nM; Kd = 0.7 nM) was a full agonist with similar potency to ET-1.