The structural interconversion between the G-quadruplex and duplex in vivo is an important subject. In the present study, we used human telomeric DNA duplex composed of GGG(TTAGGG)3/CCC(TAACCC)3 as a model system to investigate its properties under near physiological conditions by spectroscopic methods. Circular dichroism and fluorescence spectra demonstrated that G-quadruplex structure can be formed from duplex at near physiological pH (pH 7.4), salt concentration (150 mM Kþ), and temperature (37℃) in the presence of molecular crowding agent PEG (400 g/l), whereas the G-quadruplex structure cannot be formed at 25 C in buffer containing 150 mM Kþ in the presence of PEG. It is found that the formation rate of G-quadruplex structure depends on the temperature and the concentrations of both PEG and Kþ. This work suggests that human telomeric G-quadruplex structure may be potentially formed from Watson–Crick duplex in vivo.