The protein Ure2 from the yeast Saccharomyces cerevisiae has prion properties. It assembles in vitro into long, straight, insoluble fibrils that are similar to amyloids in that they bind Congo Red and show green-yellow birefringence and have an increased resistance to proteolysis. We recently showed that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta-core. A model for fibril formation, where assembly is driven by non-native inter- and/or intramolecular interaction between Ure2p monomers following subtle conformational changes was proposed [Bousset et al. (2002) EMBO J. 21, 2903-2911]. An alternative model for the assembly of Ure2p into fibrils where assembly is driven by the stacking of 40-70 N-terminal amino acid residues of Ure2p into a central beta-core running along the fibrils from which the C-terminal domains protrude was proposed [Baxa et al. (2003) J. Biol. Chem. 278, 43717-43727]. We show here that Ure2p fibril congophilia and the associated yellow-green birefringence in polarized light are not indicative that the fibrils are of amyloid nature. We map the structures of the fibrillar and soluble forms of Ure2p using limited proteolysis and identify the reaction products by microsequencing and mass spectrometry. Finally, we demonstrate that the C-terminal domain of Ure2p is tightly involved in the fibrillar scaffold using a sedimentation assay and a variant Ure2p where a highly specific cleavage site between the N- and C-terminal domains of the protein was engineered. Our results are inconsistent with the cross-beta-core model and support the model for Ure2p assembly driven by subtle conformational changes and underline the influence of the natural context of the N-terminal domain on the assembly of Ure2p.