Paramagnetic Cu(II) ions enhance nuclear spin relaxation in a distance-dependent fashion and can be used as a structural probe of proteins. Cu(II) can also serve as a functionally important ligand in proteins. Here we investigate the structural basis of Cu(II) inhibition of the influenza M2 proton channel through Cu(II)-induced paramagnetic relaxation enhancement (PRE). C-13 T-1 relaxation rates of the central residues of the transmembrane (TM) domain of M2 are significantly enhanced by Cu(II), and pronounced spectral broadening is observed for the proton-selective residue, His37. These data yielded quantitative distances of C-13 spins to the Cu(II) center and identified the Cu(II) binding site to be N epsilon 2 of His37. This binding site is surrounded by four imidazole rings from the top and four indole rings of Trp41 from the bottom, thus explaining the high affinity of Cu(II) binding. Bound at this location, Cu(II) can inhibit proton currents by perturbing histidine water proton exchange, preventing histidine conformational dynamics, and interfering with His-Trp cation-pi interaction. The Cu(II) binding site is distinct from the binding site of the hydrophobic drug amantadine, which is about 10 angstrom N-terminal to His37. Consistently, Cu(II) and amantadine induce distinct conformational changes at several key residues, suggesting the possibility of designing new drugs that target the His37 site to inhibit amantadine-resistant mutant M2 proteins. In addition to the high-affinity His37 binding site, we also examined the weaker and nonspecific binding of Cu(II) to membrane-surface lipid phosphates and the extent of the resulting PRE to surface-proximal protein residues. This study demonstrates the feasibility of NMR studies of paramagnetic-ion-complexed membrane proteins, where the ion serves as both a functional ligand and a distance probe.