High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn2+ ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H2O and in D2O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of similar to3.4 T allowed us to assign the ENDOR signals to their respective Ms manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn2+ ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein.