Two lines of transgenic mice expressing mouse/elk and mouse/horse prion protein (PrP) hybrids, which both form a well-structured β2-α2 loop in the NMR structures at 20 °C termed rigid-loop cellular prion proteins (RL-PrP(C)), presented with accumulation of the aggregated scrapie form of PrP in brain tissue, and the mouse/elk hybrid has also been shown to develop a spontaneous transmissible spongiform encephalopathy. Independently, there is in vitro evidence for correlations between the amino acid sequence in the β2-α2 loop and the propensity for conformational transitions to disease-related forms of PrP. To further contribute to the structural basis for these observations, this paper presents a detailed characterization of RL-PrP(C) conformations in solution. A dynamic local conformational polymorphism involving the β2-α2 loop was found to be evolutionarily preserved among all mammalian species, including those species for which the WT PrP forms an RL-PrP(C). The interconversion between two ensembles of PrP(C) conformers that contain, respectively, a 310-helix turn or a type I β-turn structure of the β2-α2 loop, exposes two different surface epitopes, which are analyzed for their possible roles in the still evasive function of PrP(C) in healthy organisms and/or at the onset of a transmissible spongiform encephalopathy.