NMR and fluorescence spectroscopy were used to address the effect of intracellular molecular crowding and related hydration on a model telomeric G-quadruplex (G4) DNA structure (d(AG(3)(TTAGGG)(3))). d(AG(3)(TTAGGG)(3)) prevalently adopted the hybrid-1 conformation in vivo, ex vivo, and in dilute potassium-based solution, while it formed the parallel propeller fold in water-depleted potassium-based solution, a commonly used model system for studying intracellular molecular crowding. The dilute potassium-based solution appeared to imitate the properties of the cellular environment required for d(AG(3)(TTAGGG)(3)) folding under in vivo and ex vivo conditions. High-resolution NMR investigations of site-specifically N-15-labeled G4 units in native-like single-stranded telomeric DNA revealed that the 3'-terminal and internal G4 unit predominantly coexist in 2-tetrad antiparallel basket and hybrid-2 structures that are arranged in "beads-on-a-string"-like fashion. Our data provide the first high-resolution insight into the telomeric G-overhang architecture under essentially physiological conditions and identify the 2-tetrad antiparallel basket and hybrid-2 topologies as the structural targets for the development of telomere-specific G4 ligands.