Lipophilic guanosine derivatives that form G-quadruplexes are promising building blocks for ionophores and ion channels. Herein, cation exchange between solvated cations (K+ and NH4+) and bound cations in the G-quadruplex [G1](16)center dot 4Na(+)center dot 4DNP(-) was studied by electrospray ionization mass spectrometry and solution H-1, N-15 NMR spectroscopy. The ESI-MS and 1H NMR data provided evidence for the formation of mixed-cationic Na+, K+ G(-)quadruplexes. The use of (NH4+)-N-15 cations in NMR titrations, along with N-15-filtered 1H NMR and selective NOE experiments, identified two mixed-cationic intermediates in the cation exchange pathway from [G1](16)center dot 4Na(+)center dot 4DNP(-) to [G1](16)center dot 4NH(4)(+)center dot 4DNP(-). The central Na+, bound between the two symmetry-related G(8)-Na+ octamers, exchanges with either K+ or NH4+ before the two outer Na+ ions situated within the C-4 symmetric G(8) octamers. A structural rationale, based on differences in the cations' octahedral coordination geometries, is proposed to explain the differences in site exchange for these lipophilic G-quadruplexes. Large cations such as Cs+ can be exchanged into the central cation binding site that holds the two symmetry-related C-4 symmetric G(8) octamer units together. The potential relevance of these findings to both supramolecular chemistry and DNA G-quadruplex structure are discussed.