The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP C ) form, it is a soluble, α-helix-rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, ''scrapie'' form (PrP S c ), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in β-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the α-helix-rich to the β-sheet-rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is ~7000 times more efficient than dithiothreitol.