Abstract: Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI(+)] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN(+)]. When fused to GFP and overexpressed in [ps(-)] [PIN(+)] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI(+)] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI(+)]. Two classes of gene deletions were identified. Class I deletions (bug1Delta, bem1Delta, arf1Delta, and hog1Delta) reduced the efficiency of [PSI(+)] induction, but formed rings normally. Class II deletions (las17Delta, vps5Delta, and sac6Delta) inhibited both [PSI(+)] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.