In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide bonds in Ero1p. Cys100, Cys105, Cys352, and Cys355 of Ero1p are important for oxidative protein folding and for cell viability, whereas Cys90, Cys208, and Cys349 are dispensable for these functions. Substitution of Cys100 with alanine impedes the capture of Ero1p-Pdi1p mixed-disulfide complexes from yeast, and also blocks oxidation of Pdi1p in vivo. Cys352 and Cys355 are required to maintain the fully oxidized redox state of Ero1p, and also play an auxiliary role in thiol–disulfide exchange with Pdi1p. These results suggest a model for the function of Ero1p wherein Cys100 and Cys105 form a redox-active disulfide bond that engages directly in thiol–disulfide exchange with ER oxidoreductases. The Cys352–Cys355 disulfide could then serve to reoxidize the Cys100–Cys105 cysteine pair, possibly through an intramolecular thiol–disulfide exchange reaction.