The [URE3] prion is a self-propagating amyloid form of the Ure2 protein of Saccharomyces cerevisiae. Deletions in the C-terminal nitrogen regulation domain of Ure2p increase the frequency with which the N-terminal prion domain polymerizes into the prion form, suggesting that the C-terminus stabilizes the prion domain or that the structured C-terminal region sterically impairs amyloid formation. We find by in vivo two-hybrid analysis no evidence of interaction of prion domain and C-terminal domain. Furthermore, surface plasmon resonance spectrometry shows no evidence of interaction of prion domain and C-terminal domain, and cleavage at a specific site between the domains frees the two fragments. Our NMR analysis indicates that most residues of the prion domain are in fact disordered in the soluble form of Ure2p. Deleting the tether holding the C-terminal structured region to the amyloid core does not impair prion formation, arguing against steric impairment of amyloid formation. These results suggest that the N-terminal prion domain is unstructured in the soluble protein and does not have a specific interaction with the C-terminus.