Generation of an abnormal isoform of the prion protein (PrP(Sc)) is a key aspect of the propagation of prions. Elucidation of the intracellular localization of PrP(Sc) in prion-infected cells facilitates the understanding of the cellular mechanism of prion propagation. However, technical improvement in PrP(Sc)-specific detection is required for precise analysis. Here, we show that the mAb 132, which recognizes the region adjacent to the most amyloidogenic region of PrP, is useful for PrP(Sc)-specific detection by immunofluorescence assay in cells pre-treated with guanidine thiocyanate. Extensive analysis of the intracellular localization of PrP(Sc) in prion-infected cells using mAb 132 revealed the presence of PrP(Sc) throughout endocytic compartments. In particular, some of the granular PrP(Sc) signals that were clustered at peri-nuclear regions appeared to be localized in an endocytic recycling compartment through which exogenously loaded transferrin, shiga and cholera toxin B subunits were transported. The granular PrP(Sc) signals at peri-nuclear regions were dispersed to the peripheral regions including the plasma membrane during incubation at 20 °C, at which temperature transport from the plasma membrane to peri-nuclear regions was impaired. Conversely, dispersed PrP(Sc) signals appeared to return to peri-nuclear regions within 30 min during subsequent incubation at 37 °C, following which PrP(Sc) at peri-nuclear regions appeared to redisperse again to peripheral regions over the next 30 min incubation. These results suggest that PrP(Sc) is dynamically transported along with the membrane trafficking machinery of cells and that at least some PrP(Sc) circulates between peri-nuclear and peripheral regions including the plasma membrane via an endocytic recycling pathway.