The conversion of the normal cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP(C) and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP(C) we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP(C) and on PrP(Sc) formation. We found that ER-targeted anti-prion scFvs retain PrP(C) in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP(C), with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP(C) acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP(Sc) accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.