PrP106 -126, a peptide corresponding to residues 107-127 of the human prion protein, induces neuronal cell death by apoptosis and causes proliferation and hypertrophy of glia, reproducing the main neuropathological features of prion-related transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Although PrP106 -126 has been shown to form amyloid-like fibrils in vitro, their structural properties have not been elucidated. Here, we investigate the conformational characteristics of a fibril-forming fragment of the mouse prion protein, MoPrP106 -126, by using electron microscopy, CD spectroscopy, NMR-detected hydrogen-deuterium exchange measurements, and molecular dynamics simulations. The fibrils contain Ϸ50% ␤-sheet structure, and strong amide exchange protection is limited to the central portion of the peptide spanning the palindromic sequence VAGAAAAGAV. Molecular dynamics simulations indicate that MoPrP106 -126 in water assumes a stable structure consisting of two four-stranded parallel ␤-sheets that are tightly packed against each other by methyl-methyl interactions. Fibril formation involving polyalanine stacking is consistent with the experimental observations. P rions are infectious particles that cause transmissible spongiform encephalopathies in animals and humans. Prions are composed of PrP Sc , a conformationally altered form of a hostencoded glycoprotein, PrP C (1). Although the two isoforms are chemically identical, they possess very different physicochemical properties. In particular, PrP C is mostly helical, whereas the scrapie form PrP Sc contains Ϸ40% ␤-sheet (2). A synthetic peptide, PrP106-126, was shown to aggregate into proteaseresistant amyloid fibrils and induce neuronal cell death by apoptosis, causing proliferation and hypertrophy of cultured glia (3, 4). This segment corresponds to an unstructured region just outside of the globular C-terminal domain of PrP C (5, 6). Analysis of deletion mutants of human prion protein (PrP) showed that a large N-terminal fragment (residues 23-88) and a segment within the structured domain of PrP C (residues 141-176) could be deleted without affecting its conversion into the protease-resistant PrP Sc , whereas deletion of segments 95-107, 108-121, or 122-140 abolished the conformational transition (7). PrP106-126 is located within this critical region (residues 95-140), has been shown to adopt different secondary structures under different solution conditions (8, 9), and is thus a relevant model for investigating the mechanism of fibril formation and PrP Sc -mediated cell death. Recent solid-state NMR results showed that fibrils of the mouse prion peptide 89-143 are composed predominantly of ␤-structure, and they suggested that its pathogenicity is related to the specific ␤-sheet conformation (10, 11). However, little is known presently about the detailed structure of PrP Sc or any fibril-forming fragments of the PrP. In fact, there are very few direct experimental observations available for any...