The structure of a stable model protein, bovine pancreatic trypsin inhibitor (BPTI), in the lyophilized form has been investigated using the hydrogen isotope exchange/high-resolution NMR methodology. Six amide protons of BPTI that are buried in the protein interior and strongly hydrogen-bonded in aqueous solution are found to exchange with water vapors within hours in the lyophilized state; in aqueous solution, most of these protons do not exchange appreciably even after a week under otherwise identical conditions. When BPTI is lyophilized in the presence of the lyoprotectant sorbitol, no significant hydrogen isotope exchange of these protons in the solid state is detected. On the basis of these and other observations it is concluded that the structure of BPTI is partially (and reversibly) denatured on lyophilization. This conclusion, if true for other proteins, may explain the drastically reduced enzymatic activity in nonaqueous media compared to that in water.