A recombinant protein corresponding to the human prion protein domain encompassing residues 90-231 (huPrP(90-231)) was expressed in Escherichia coli in a soluble form and purified to homogeneity. Spectroscopic data indicate that the conformational properties and the folding pathway of huPrP(90-231) are strongly pH-dependent. Acidic pH induces a dramatic increase in the exposure of hydrophobic patches on the surface of the protein. At pH between 7 and 5, the unfolding of hPrP(90-231) in guanidine hydrochloride occurs as a two-state transition. This contrasts with the unfolding curves at lower pH values, which indicate a three-state transition, with the presence of a stable protein folding intermediate. While the secondary structure of the native huPrP(90-231) is largely α-helical, the stable intermediate is rich in β-sheet structure. These findings have important implications for understanding the initial events on the pathway toward the conversion of the normal into the pathological forms of prion protein.