The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated h-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D 1 H NMR in order to assess the extent of assignments allowed by 1 H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the h-chain to the crystallographically characterized complexes that contains the h-chain. The target residues are those that exhibit significant (> | 0.2| ppm) dipolar shifts, as predicted by a ''preliminary'' set of magnetic axes determined from a small set of easily assigned active site residues. All 104 target residues (f 70% of total) were assigned by taking advantage of the temperature dependence predicted by the ''preliminary'' magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than | 0.2| ppm. These comprehensive assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the h-chain in terms of the crystal coordinates of the h-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated h-chain relative to that in the intact R-state HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted f 10j from the heme normal so that the Fe-CN unit is tilted toward the h-meso-H in a fashion remarkably similar to the Fe-CO tilt in the h-subunit of HbCO. It is concluded that a set of ''preliminary'' magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet h-chain and that this approach should be similarly effective in HbA.