The complexes formed by kanamycin A at three different pH values (5.5, 7.4 and 12.0) were investigated by NMR and EPR spectroscopy. Paramagnetic relaxation contributions to proton relaxation rates were measured using a combination of the TOCSY sequence with the inversion recovery experiment in order to gain signal resolution in the bulk region. Measured contributions were converted into distances and used for structural determination by restrained simulated annealing where all possible chair and boat conformations of the rings were taken into account. The interaction of the Cu(II) ion with the nitrogen of the C ring is apparent at all pH values. At higher pH also the amino group of ring A starts to be involved in the metal coordination sphere. This is accompanied by a switch in conformation of ring C. Structures are consistent with the involvement in the coordination sphere either of the 2′ or 4′ hydroxyl oxygens at pH 5.5 and the 5 and the 6′ hydroxyl oxygens (or the ring oxygen) at pH 12.0.