We have reported that human protein disulfide isomerase-related protein (hPDIR) has isomerase and chaperone activities that are lower than those of the human protein disulfide isomerase (hPDI), and that the b domain of hPDIR is critical for its chaperone activity [J. Biol. Chem. 279 (2004) 4604]. To investigate the basis of the differences between hPDI and hPDIR, and to determine the functions of each hPDIR domain in detail, we constructed several hPDIR domain mutants. Interestingly, when the b domain of hPDIR was replaced with the b' domain of hPDI, a dramatic increase in chaperone activity that was close to that of hPDI itself was observed. However, this mutant showed decreased oxidative refolding of alpha1-antitrypsin. The replacement of the b domain of hPDIR with the c domain of hPDI also increased its chaperone activity. These observations suggest that putative peptide-binding sites of hPDI determine both its chaperone activity and its substrate specificity.