G-Rich oligonucleotides with cytosine residues in their sequences can form G-quadruplexes where G-quartets are flanked by G·C Watson-Crick base pairs. In an attempt to probe the role of cations in stabilization of a structural element with two G·C base pairs stacked on a G-quartet, we utilized solution state nuclear magnetic resonance to study the folding of the d(G(3)CT(4)G(3)C) oligonucleotide into a G-quadruplex upon addition of (15)NH(4)(+) ions. Its bimolecular structure exhibits antiparallel strands with edge-type loops. Two G-quartets in the core of the structure are flanked by a couple of Watson-Crick G·C base pairs in a sheared arrangement. The topology is equivalent to the solution state structure of the same oligonucleotide in the presence of Na(+) and K(+) ions [Kettani, A., et al. (1998) J. Mol. Biol.282, 619, and Bouaziz, S., et al. (1998) J. Mol. Biol.282, 637). A single ammonium ion binding site was identified between adjacent G-quartets, but three sites were expected. The remaining potential cation binding sites between G-quartets and G·C base pairs are occupied by water molecules. This is the first observation of long-lived water molecules within a G-quadruplex structure. The flanking G·C base pairs adopt a coplanar arrangement and apparently do not require cations to neutralize unfavorable electrostatic interactions among proximal carbonyl groups. A relatively fast movement of ammonium ions from the inner binding site to bulk with the rate constants of 21 s(-1) was attributed to the lack of hydrogen bonds between adjacent G·C base pairs and the flexibility of the T(4) loops.