Hydrogen bonds stabilize protein and nucleic acid structure, but little direct spectroscopic data have been available for characterizing these critical interactions in biological macromolecules. It is demonstrated that the electric field gradient at the nucleus of an amide hydrogen can be determined residue-specific by measurement of 15N NMR relaxation times in proteins dissolved in D2O, and uniformly enriched with 13C and 15N. In D2O, all backbone amide protons can be exchanged with solvent deuterons, and the T1 relaxation rate of a deuteron is dominated by its quadrupole coupling constant (QCC), which is directly proportional to the electric field gradient at the nucleus. 2HN T1 relaxation can be measured quantitatively through its effect on the T2 relaxation of its directly attached 15N. QCC values calculated from 2HN T1 and previously reported spectral densities correlate with the inverse cube of the X-ray crystal structure-derived hydrogen bond lengths: QCC = 228 + Sigmai 130 cos alphai/ri3 kHz, where alpha is the N-H...Oi angle and r is the backbone-backbone (N-)H...Oi(=C) hydrogen bond distance in ângstroms.