The protein Ure2 from the yeast Saccharomyces cerevisiae has prion properties. In vitro and at neutral pH, soluble Ure2p spontaneously forms long, straight, insoluble protein fibrils. Two models have been proposed to account for the assembly of Ure2p into protein fibrils. The “amyloid backbone” model postulates that a segment ranging from 40 to 70 amino acid residues from the flexible N-termini of Ure2p form a parallel superpleated ?-structure running along the fibrils (Kajava et al, (2004) Proc Natl Acad Sci USA 101: 7885-7890). The second model hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and/or intra-molecular interactions between Ure2p monomers (Bousset et al, (2002) EMBO J 21: 2903-2911). Here we perform a cystein scan on residues located in the N- and C-terminal parts of Ure2p to determine whether these domains interact. We show that amino acid stretches centered around residues 6, in the N-terminal domain of Ure2p, and 137, in the C-terminal moiety, interact at least transiently via intra-molecular interactions. We document the assembly properties of the Ure2p variant where a disulfide bond is established between the N- and C- terminal domains and show it possesses assembly properties indistinguishable from that of wild type Ure2p. We probe the structure of Ure2pC6C137 within the fibrils and demonstrate that the polypeptide is in a conformation similar to that of its soluble assembly-competent state. Our results constitute the first structural characterization of the N-terminal domain of Ure2p in both its soluble assembly competent and fibrillar forms. Our data indicate that the flexibility of the N-terminal domain and conformational changes within this domain are essential for fibril formation and provide new insight into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] phenotype in yeast.