Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM(2) (1-4). The mutations are predicted to block folding of the precursor in the ER of pancreatic beta-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5-7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired beta-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11-13) and the structural basis of disulfide pairing (14-19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.