According to the “protein-only” hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal (PrP^C) and pathological (PrP^Sc) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90−231 of the human prion protein (huPrP90−231). Incubation of the protein under acidic conditions (pH 3.6−5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an α-helical conformation to a β-sheet structure and oligomerization of huPrP90−231 into large molecular weight aggregates. No stable monomeric β-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of β-sheet structure and protein oligomerization likely occur concomitantly. The β-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrP^Sc). Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP^Sc-like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90−231 to an oligomeric β-sheet structure is the presence of salt.