Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.