Absolute assignments were made of the side-chain amide proton and nitrogen resonances of a series of cobalamins (Cbls, derivatives of vitamin and two cobinamides [ Cbis, analogs in which the axial 5,6-B 12 ) dimethylbenzimidazole (Bzm) ligand has been chemically removed ] in water. The syn and anti amide protons could always be distinguished based on the magnitudes of their NOEs to the neighboring methylene protons in ROESY spectra. The chemical shifts of the c amide nitrogen and anti proton of the two aqua complexes, aquacobalamin and 10-chloroaquacobalamin, are signiÐcantly perturbed from those of the other complexes and demonstrate that a hydrogen bond between the c carbonyl and the coordinated water molecule, previously seen in the x-ray crystal structures of these complexes, persists in aqueous solution. The 15N chemical shift of the secondary f amide in the nucleotide loop of the CblÏs correlates linearly (r2 = 0.997) with the solid-state axial CoÈN bond distance, providing an excellent NMR probe of this bond length. Removal of the axial Bzm to form the CbiÏs results in changes in the amide nitrogen and proton chemical shifts of the "downwardlyÏ projecting side-chains, characteristic of the inÑuence of the magnetic anisotropy of the Bzm moiety on these amides in the Cbl complexes. Amide proton chemical shift thermal gradients and 15N solvent-dependent chemical shifts are interpreted as being due to complex patterns of intramolecular hydrogen-bonded side-chain amides in equilibrium with forms fully hydrogen bonded to solvent.