A solution state NMR study has shown that d(G(4)T(3)G(4)) in the presence of (NH4+)-N-15 ions folds into a single bimolecular G-quadruplex structure in which its G-tracts are antiparallel and the two T-3 loops span along the edges of the outer G-quartets on the opposite sides of the G-quadruplex core. This head-to-tail topology is in agreement with the topology of the G-quadruplex recently found in the X-ray crystal structure formed by d(G(4)T(3)G(4)) in the presence of K+ ions [Neidle et al. J. Am. Chem. Soc. 2006, 128, 5480]. In contrast, the presence of K+ ions in solution resulted in a complex ensemble of G-quadruplex structures. Molecular models based on NMR data demonstrate that thymine loop residues efficiently base-base stack on the outer G-quartets and in this way stabilize a single structure in the presence of (NH4+)-N-15 ions. The use of heteronuclear NMR enabled us to localize three (NH4+)-N-15 ion binding sites between pairs of adjacent G-quartets and study the kinetics of their movement. Interestingly, no (NH4+)-N-15 ion movement within the G-quadruplex was detected at 25 degrees C. At 35 degrees C we were able to observe slow movement of (NH4+)-N-15 ions from the outer binding sites to bulk solution with the characteristic residence lifetime of 1.2 s. The slow movement of (NH4+)-N-15 ions from the outer binding sites into bulk solution and the absence of movement from the inner binding site were attributed to steric hindrance imposed by the T-3 loops and the rigidity of the G-quadruplex.