Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.