Calnexin and calreticulin interact specifically with newly synthesized glycoproteins in the endoplasmic reticulum (ER) and function as molecular chaperones. The carbohydrate-specific interactions between ER components and glycoproteins synthesized in isolated canine pancreatic microsomes were analyzed using a cross-linking approach. A carbohydrate-dependent interaction between newly synthesized glycoproteins, the thiol-dependent reductase ERp57, and either calnexin or calreticulin was identified. The interaction between ERp57 and the newly synthesized glycoproteins required trimming of the N-linked oligosaccharide side chain. Thus, it is likely that ERp57 functions as part of the glycoprotein-specific quality control machinery operating in the lumen of the ER.