Prions are proteins that can adopt different infectious conformations known as "strains" or "variants," each with a distinct, epigenetically inheritable phenotype. Mechanisms by which prion variants are determined remain unclear. Here we use the Saccharomyces cerevisiae prion Rnq1p/[PIN(+)] as a model to investigate the effects of chaperone proteins upon prion variant determination. We show that deletion of specific chaperone genes alters [PIN(+)] variant phenotypes, including [PSI(+)] induction efficiency, Rnq1p aggregate morphology/size and variant dominance. Mating assays demonstrate that gene deletion-induced phenotypic changes are stably inherited in a non-Mendelian manner even after restoration of the deleted gene, confirming that they are due to a bona fide change in the [PIN(+)] variant. Together, our results demonstrate a role for chaperones in regulating the prion variant complement of a cell.