The formation of guanine quartets with 9-isopropylguanine (ⁱPG) is discussed in organic solvents. Crystal structures of the ⁱPG quartets were determined by X-ray crystallography with template cations (Na⁺ and Ca²⁺) and the structure without a template cation was also obtained by virtue of the stabilization by intermolecular hydrogen bonding with water molecules of crystallization. The difference in the quartet formation of ⁱPG in the presence and absence of a template cation was clearly demonstrated by ¹H NMR measurements in CDCl₃–CH₃OH mixed solvents. The quartet formation is mainly governed by the enthalpy gain due to the electrostatic interaction between the O6 oxygen in ⁱPG and the template cations in the presence of the cations rather than the intermolecular hydrogen bonding, while desolvation of ⁱPG is the dominant factor for the formation in the absence of cations. In the presence of Na⁺ and Ca²⁺, ΔH and ΔS values in the formation of ⁱPG-4–Na⁺ and ⁱPG-4–Ca²⁺ complexes were determined to be ΔH = −8.4 kcal mol⁻¹ and ΔS = +50 cal mol⁻¹ K⁻¹ for Na⁺ and ΔH = −12.9 kcal mol⁻¹ and ΔS = +34 cal mol⁻¹ K⁻¹ for Ca²⁺ on the basis of van't Hoff plots attained from the results of temperature-dependent UV-Vis spectroscopic measurements.