Yeast prions are inherited through proteins that exist in alternate, self-perpetuating conformational states. The mechanisms by which these states arise and are maintained are still poorly defined. Here we demonstrate for the first time that Sis1, a member of the Hsp40 chaperone family, plays a critical role in the maintenance of a prion. The prion [RNQ+] is formed by Rnq1, which is present in the same physical complex as Sis1, but only when Rnq1 is in the prion state. The G/F domain of Sis1 is dispensable for rapid growth on rich medium, but is required for [RNQ+] maintenance, distinguishing essential regions of Sis1 from those needed for prion interaction. A specific Sis1 deletion mutant altered the physical aggregation pattern of Rnq1 without curing the prion. This variant state propagated in a heritable fashion after wild-type Sis1 function was restored, indicating that multiple physical states are compatible with prion maintenance and that changes in chaperone activity can create prion variants. Using a prion chimera we demonstrate that the prion-determinant domain of Rnq1 is genetically sufficient for control by Sis1.