The [ PSI ⁺ ] determinant of Saccharomyces cerevisiae , consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers. The molecular chaperone Hsp104 is required to maintain self-replication of [ PSI ⁺ ]. We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup35 ^5–26 , results in the dissociation of oligomeric Sup35 into monomeric species. Several intermediates of Sup35 ^5–26 could be detected during this process. Strong interactions are found between Hsp104 and hexameric/tetrameric Sup35 ^5–26 , whereas the intermediate and monomeric “release” forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments. Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup35 ^5–26 . No interaction can be detected between Hsp104 and higher oligomeric states (≥8) of Sup35 ^5–26 . Taking into account the fact that Hsp104 is required for maintenance of [ PSI ⁺ ], we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast.