It has previously been shown that yeast prion [PSI+] is cured by GuHCl, although reports on reversibility of curing were contradictory. Here we show that GuHCl treatment of both [PSI+] and [psi-] yeast strains results in two classes of [psi-] derivatives: Pin+, in which [PSI+] can be reinduced by Sup35p overproduction, and Pin-, in which overexpression of the complete SUP35 gene does not lead to the [PSI+] appearance. However, in both Pin+ and Pin- derivatives [PSI+] is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, [PSI+] curing remains reversible in both cases. Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin+ [psi-] derivatives are observed in Pin- [psi-] derivatives. Genetic analyses show that the Pin+ phenotype is determined by a non-Mendelian factor, which, unlike the [PSI+] prion, is independent of the Sup35p N-terminal domain. A Pin- [psi-] derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while [PSI+] curing by Hsp104 overproduction resulted exclusively in Pin+ [psi-] derivatives. We hypothesize that in addition to the [PSI+] prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin+ phenotype.