Protein-disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes dithiol oxidation and disulfide bond reduction and isomerization using the active site CGHC. Haploid pdi1 delta Saccharomyces cerevisiae are inviable, but can be complemented with either a wild-type rat PDI gene or a mutant gene coding for CGHS PDI (shufflease). In contrast, pdi1 delta yeast cannot be complemented with a gene coding for SGHC PDI. In vitro, shufflease is an efficient catalyst for the isomerization of existing disulfide bonds but not for dithiol oxidation or disulfide bond reduction. SGHC PDI catalyzes none of these processes. These results indicate that in vivo protein folding pathways contain intermediates with non-native disulfide bonds, and that the essential role of PDI is to unscramble these intermediates.