Upon nonreducing Tris-Tricine-urea-SDS-PAGE, newly synthesized proinsulin from pancreatic islets of normal rodents forms a band fast mobility representing the native disulfide isomer, which is efficiently secreted. In addition at least two slower migrating "isomer 1 and 2" bands are recovered, not discernible under reducing conditions, which represent minor species that exhibit less efficient secretion. Although rats and mice have two proinsulin genes, three distinct migrating species are also produced upon proinsulin expression from a single wildtype human proinsulin cDNA. The "Akita-type" proinsulin mutation, which causes dominant-negative diabetes mellitus due to point mutation C(A7)Y that leaves B7-cysteine without its disulfide pairing partner, is recovered as a form that near quantitatively co-migrates with the aberrant isomer 1 band of proinsulin. Anomalous migration is also demonstrated for several other mutants lacking a single cysteine. In islets from PERK-/- mice, which exhibit premature loss of pancreatic beta cells, hypersynthesis of proinsulin increases the amount of nonnative proinsulin isomers. Such findings appear consistent with an hypothesis that supranormal production of nonnative proinsulin may predispose to pancreatic beta cell toxicity.