Prion diseases are fatal and transmissible neurodegenerative disorders characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP(C)) denoted PrP(Sc). To identify intracellular organelles involved in PrP(Sc) formation, we studied the role of the Ras-related GTP-binding proteins Rab4 and Rab6a in intracellular trafficking of the prion protein and production of PrP(Sc). When a dominant-negative Rab4 mutant or a constitutively active GTP-bound Rab6a protein was overexpressed in prion-infected neuroblastoma N2a cells, there was a marked increase of PrP(Sc) formation. By immunofluorescence and cell fractionation studies, we have shown that expression of Rab6a-GTP delocalizes PrP within intracellular compartments, leading to an accumulation in the endoplasmic reticulum. These results suggest that prion protein can be subjected to retrograde transport toward the endoplasmic reticulum and that this compartment may play a significant role in PrP(Sc) conversion.