As a model for understanding how protein disulfide isomerase (PDI) catalyzes disulfide bond formation in proteins, its action on a 28-residue disordered peptide containing only two cysteine residues has been examined. Disulfide formation in the peptide using the chemical reaction with small molecule thiol/disulfide reagents, such as oxidized and reduced glutathione or cystamine and cysteamine, occurs in two steps, via two alternative intermediate mixed disulfides between the reagent and either peptide cysteine residue. All thiol/disulfide forms of the peptide could be trapped and quantified, so the rates of their interconversion could be measured. Catalytic amounts of PDI increased the rates of these reactions. All rate enhancements were independent of the concentration of the peptide, indicating that it bound to PDI with an apparent K-m of less than 3 mu M. In the presence of glutathione, PDI accelerated the formation of both single mixed disulfide species, plus their subsequent rearrangement to form the peptide disulfide bond, but not interchange of the mixed disulfide glutathione between the two cysteine residues. In contrast, PDI did not catalyze the reaction of the reagent cystamine with the reduced peptide to form the mixed disulfide, nor the interchange of this mixed disulfide between cysteine residues, but it did catalyze the subsequent intramolecular step of peptide disulfide bond formation to a similar extent as with the glutathione mixed disulfide. These effects on the two steps involving the mixed disulfides with glutathione or cystamine accounted for much of the overall catalytic effect of PDI on disulfide bond formation in the peptide, indicating that direct transfer of disulfide bonds from PDI to the peptide occurred less frequently. These findings demonstrate the utility of using such peptides as PDI substrates and have implications for the mechanism of action of PDI