Background: Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPC. The conversion and thus the propensity of PrPC to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physicochemical properties of proteins as well as the dynamics and structure of folding intermediates. Results: Conformational intermediates of the human prion protein huPrP(C) were characterized by a combination of hydrostatic pressure (up to 200 MPa) with two-dimensional NMR spectroscopy. All pressure effects showed to be reversible and there is virtually no difference in the overall pressure response between the folded core of the N-terminal truncated huPrP(C)(121-230) and the full-length huPrP(C)(23-230). The only significant differences in the pressure response of full-length and truncated PrP suggest that E168, H187, T192, E207, E211 and Y226 are involved in a transient interaction with the unfolded N-terminus. High-pressure NMR spectroscopy indicates that the folded core of the human prion protein occurs in two structural states N-1 and N-2 in solution associated with rather small differences in free enthalpies (3.0 kJ/ mol). At atmospheric pressure approximately 29% of the protein are already in the pressure favored conformation N-2. There is a second process representing two possible folding intermediates I-1 and I-2 with corresponding average free enthalpies of 10.8 and 18.6 kJ/ mol. They could represent preaggregation states of the protein that coexist at ambient pressure with a very small population of approximately 1.2% and less than 0.1%. Further the pressure response of the N-terminus indicates that four different regions are in a fast equilibrium with non-random structural states whose populations are shifted by pressure. Conclusion: We identified pressure stabilized folding intermediates of the human prion protein. The regions reflecting most strongly the transition to the intermediate states are the beta 1/alpha 1-loop and the solvent exposed side of alpha 3. The most pressure-sensitive region (representing mainly intermediate I-1) is the loop between beta-strand 1 and alpha-helix 1 (residue 139-141), indicating that this region might be the first entry point for the infectious conformer to convert the cellular protein.