Earlier we showed altered expression of Hsp40s, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain proteins affect prion propagation in wild type and Hsp70 mutant cells. We also identified several Hsp40 and TPR protein mutant alleles that impair or enhance prion propagation. Our data suggest that many, if not all, of the observed effects of co-chaperones on prions are mediated by their regulation of Hsp70 activities. In many instances the same conditions producing a significant effect on one prion have either little or an opposite effect on a different prion. These data point to a prion preference or specificity of the Hsp70/co-chaperone pairings. Targeting the two major yeast Hsp40s (Ydj1p and Sis1p), we have identified mutations that alter prion propagation in both positive and negative ways. We showed that elimination of the URE3 prion by overproduced Ydj1p requires only its ability to regulate Hsp70, which demonstrated that "curing" was indirect and required interaction of Hsp40 with Hsp70, but not amyloid. We also showed that while the intact Sis1p is unable to cure cells of URE3, the Hsp70-regulating J-domain of Sis1p could do so when separated from the remainder of the protein, highlighting both specificity of Hsp40 regulation of Hsp70 and functional redundancy of Hsp40 subdomains. We also showed that curing efficiency depended on the particular isoform of Hsp70. We recently identified certain activities of Sis1p that specifically affect the ability of a mutant Hsp70 to interfere with propagation of the PSI+ prion. Similar alterations of Sis1p have no detectable effect on prions in wild type cells. These findings show that the mutant Hsp70 depends on Sis1p in order for it to exert its anti-prion effects, and that several Hsp40 activities of Sis1p are dispensable for prion propagation. Therefore, propagation and elimination of prions require distinct chaperone activities, and Hsp40s other than Sis1p must provide the Hsp40 activities required for prion replication. The latter finding contradicts earlier proposals that Sis1p is essential for PSI+ propagation and is critical for recruiting Hsp70 and Hsp104 to prion polymers in vivo. When cells express these mutated Sis1 proteins in place of wild type Sis1p, overexpressing Hsp104, which normally causes cells to lose PSI+ prions, has no effect on them. These results show that while Hsp40s other than Sis1p can contribute to the prion replication mechanism, Sis1p is required as a specific Hsp40 for the Hsp104-mediated curing of prions. These findings are consistent with prion propagation and elimination requiring different chaperone activities. They also confirm a link between the anti-prion effects of specifically altered Hsp70 function and overexpressed Hsp104 and suggest that altering Hsp70 in specific ways can inhibit prions by the same mechanism as increasing abundance of Hsp104.