Iron dyshomeostasis has been observed in prion diseases; however, little is known regarding the contribution of the oxidation state of iron to prion protein (PrP) conversion. In this study, PrPC-deficient HpL3-4 cells were exposed to divalent [Fe(II)] or trivalent [Fe(III)] iron, followed by exogenous recombinant PrP (rPrP) treatment. We then analyzed the accumulation of internalized rPrP and its biochemical properties, including its resistance to both proteinase K (PK) digestion and detergent solubility. Fe(III), but not Fe(II), induced the accumulation of internalized rPrP, which was partially converted to detergent-insoluble and PK-resistant PrP (PrPres). The Fe(III)-induced PrPres generation required an intact cell structure, and it was hindered by U18666A, an inhibitor of vesicular trafficking, but not by NH4Cl, an inhibitor of endolysosomal acidification. These observations implicated that the Fe(III)-mediated PrPres conversion likely occurs during endosomal vesicular trafficking rather than in the acidic environment of lysosomes. (C) 2013 Elsevier Inc. All rights reserved.