The prion protein (PrP) possesses sequence-specific domains that endow the molecule with neuroprotective and neurotoxic activities, and that may contribute to the pathogenesis of prion diseases. To further define critical neurotoxic determinants within PrP, we previously generated Tg(DeltaCR) mice that express a form of PrP harboring a deletion of 21 amino acids within the central domain of the protein [Li et al., EMBO J. 26 (2007), 548]. These animals exhibit a neonatal lethal phenotype that is dose-dependently rescued by co-expression of wild-type PrP. In this study, we examined the localization and cell biological properties of the PrP(DeltaCR) protein in cultured cells to further understand the mechanism of PrP(DeltaCR) neurotoxicity. We found that the distribution of PrP(DeltaCR) was identical to that of wild-type PrP in multiple cell lines of both neuronal and non-neuronal origin, and that co-expression of the two proteins did not alter the localization of either one. Both proteins were found in lipid rafts, and both were localized to the apical surface in polarized epithelial cells. Taken together, our results suggest that PrP(DeltaCR) toxicity is not a result of mislocalization or aggregation of the protein, and more likely stems from altered binding interactions leading to the activation of deleterious signaling pathways.