The Sup35 protein can exist in a non-infectious form or in various infectious forms called [PSI+] prion variants (or prion strains). Each of the different [PSI+] prion variants converts non-infectious Sup35 molecules into that prion variant's infectious form. One definition of a ‘prion domain’ is the minimal fragment of a prion protein that is necessary and sufficient to maintain the prion form. We now demonstrate that the Sup35 N region (residues 1–123), which is frequently referred to as the ‘prion domain’, is insufficient to maintain the weak or strong [PSI +] variants per se, but appears to maintain them in an ‘undifferentiated’[PSI+] state that can differentiate into weak or strong [PSI+] variants when transferred to the full-length Sup35 protein. In contrast, Sup35 residues 1–137 are necessary and sufficient to faithfully maintain weak or strong [PSI+] variants. This implicates Sup35 residues 124–137 in the variant-specific maintenance of the weak or strong [PSI+] forms. Structure predictions indicate that the residues in the 124–137 region form an α-helix and that the 1–123 region may have β structure. In view of these findings, we discuss a plausible molecular basis for the [PSI+] prion variants as well as the inherent difficulties in defining a ‘prion domain’.