This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5'-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K and Na). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.Communicated by Ramaswamy H. Sarma.