Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrP(Sc), a disease-associated isoform of the host-encoded cellular protein (PrP(C)). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc). However, PrP(Sc) is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrP(Sc) aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C) and PrP(Sc) by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrP(Sc) solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl](1/2) values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl](1/2) values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrP(Sc), we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc) aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrP(Sc) conformational stabilities of protease-resistant and protease-sensitive PrP(Sc) and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.