THE conversion of normal, protease sensitive prion protein (PrP-sen) to the abnormal protease-resistant form (PrP-res) is of central importance in the pathogenesis of scrapie and other transmissible spongiform encephalopathies. In the present study, the effects of reduction of the disulfide bond on the PrP-sen to PrP-res conversion in a cell-free system were examined. The addition of the disulfide reducing agent dithiothreitol inhibited the cell-free conversion reaction with an IC50of 2–2.5 mM. Separate pretreatment of either PrP-sen or PrP-res with dithiothreitol and an alkylating agent also inhibited the conversion reaction. Results of this study show that preservation of the disulfide bond is important in the conversion of PrP-sen to PrP-res.