The mammalian prion protein can form infectious, nonnative, and protease resistant aggregates (PrP), which cause lethal prion diseases like human Creutzfeldt-Jakob disease. PrP seeds the formation of new infectious prions by interacting with and triggering the refolding of the normally soluble mammalian prion protein, PrP, into more PrP. Refolding of misfolded proteins in the cell is carried out by molecular chaperones such as Grp78. We have recently shown that Grp78 sensitizes PrP to proteases, indicating structural alterations and leading to its degradation. However, the process of chaperone-mediated PrP disaggregation, the chaperones involved, and the effect of disaggregation on PrP seeding activity are unclear. We have now monitored the structural modification, disaggregation, and seeding activity of PrP from two mouse adapted prion strains, 22L and 87V, in the presence of Grp78 and two forms of the Hsp110 disaggregase chaperone family, Hsp105 and Apg-2. We found that both forms of Hsp110 induced similar amounts of disaggregation and structural change in the protease resistant cores of PrP from both strains. However, 22L PrP was more susceptible to destabilization and disaggregation by the chaperones than 87V. Surprisingly, despite disaggregation of both strains, only the 22L PrP aggregates released by the chaperones had seeding activity, with both forms of Hsp110 enhancing the Grp78 mediated release of these aggregates. Our data show that disassembly of PrP by Grp78 and Hsp110 chaperones can release seeding particles of PrP in a strain-specific manner, potentially facilitating prion replication and spread.