Major histocompatibility complex (MHC) class I molecules bind and present short peptides to cells of the immune system. The oxidoreductase ERp57 is involved in the assembly of MHC class I molecules and is a component of the peptide loading complex, where it is found disulfide-bonded to tapasin. We have studied ERp57 and the ERp57-tapasin conjugate by rapid acidification of the intracellular environment with trichloroacetic acid (TCA), followed by thiol modification with the alkylating agent 4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). By using TCA/AMS treatment, non-tapasin-associated ERp57 is shown to exist almost exclusively in a reduced state, suggesting that both thioredoxin-like CXXC motifs are exposed and reduced. A 110-kDa product is readily detected with this TCA/AMS protocol and is confirmed as an ERp57-tapasin conjugate by its absence from the tapasin-deficient .220 cell line and by immunoblotting with both ERp57- and tapasin-specific antisera. The ERp57-tapasin conjugate can also be modified with the oxidizing agent diamide, indicating that within the pool of ERp57-tapasin complexes the free, non-tapasin-linked CXXC motif exists in both oxidized and reduced states, suggesting availability to undergo redox reactions.