The recombinant murine prion protein, mPrP(23–231), was expressed in E. coli with uniform ¹⁵N-labeling. NMR experiments showed that the previously determined globular three-dimensional structure of the C-terminal domain mPrP(121–231) is preserved in the intact protein, and that the N-terminal polypeptide segment 23–120 is flexibly disordered. This structural information is based on nearly complete sequence-specific assignments for the backbone amide nitrogens, amide protons and α-protons of the polypeptide segment of residues 121–231 in mPrP(23–231). Coincidence of corresponding sequential and medium-range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121–231) are also present in mPrP(23–231), and near-identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three-dimensional fold of mPrP(121–231) is also preserved in the intact protein. The linewidths in heteronuclear ¹H–¹⁵N correlation spectra and ¹⁵N{¹H}-NOEs showed that the well structured residues 126–230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23–231) outside of this domain.