The folding and maturation of most secreted proteins requires the formation of disulphide bonds, which are introduced co- and post-translationally in the endoplasmic reticulum. Disulphide bonds form by a redox relay that involves protein disulphide isomerases (PDIs) and oxidases, and a terminal electron acceptor, which, in vitro, has been shown to be oxygen. Koritzinsky et al. now find that disulphide bond formation in mammalian cells occurs in two phases — a first phase that does not require oxygen, and a second (post-translational) phase that does require oxygen. This suggests that early co-translational disulphide bond formation uses different PDIs and oxidases, which remain to be identified, as well as their alternative electron acceptor. Furthermore, it indicates that post-translational oxygen-dependent disulphide bond formation is the underlying cause for hypoxia being a trigger of the unfolded protein response.