Our concept of how disulfides form in proteins entering the secretory pathway has changed dramatically in recent years. The discovery of endoplasmic reticulum (ER) oxidoreductin 1 (ERO1) was followed by the demonstration that this enzyme couples oxygen reduction to de novo formation of disulfides. However, mammals deficient in ERO1 survive and form disulfides, which suggests the presence of alternative pathways. It has recently been shown that peroxiredoxin 4 is involved in peroxide removal and disulfide formation. Other less well-characterized pathways involving quiescin sulfhydryl oxidase, ER-localized protein disulfide isomerase peroxidases and vitamin K epoxide reductase might all contribute to disulfide formation. Here we discuss these various pathways for disulfide formation in the mammalian ER and highlight the central role played by glutathione in regulating this process.