Formation of DNA quadruplexes requires monovalent cation binding. To characterize the cation binding stoichiometry and linkage between binding and folding, we carried out KCl titrations of Tel22 (d[A(GGGTTA)(3)]), a model of the human telomere sequence, using a fluorescent indicator to determine [K(+)](free) and circular dichroism to assess the extent of folding. At [K(+)](free)=5 mM (sufficient for >95% folding), the apparent binding stoichiometry is 3K(+)/Tel22; at [K(+)](free)=20 mM, it increased to 8-10K(+)/Tel22. Thermodynamic analysis shows that at [K(+)](free)=5 mM, K(+) binding contributes approximately -4.9 kcal/mol for folding Tel22. The overall folding free energy is -2.4 kcal/mol, indicating that there are energetically unfavorable contributions to folding. Thus, quadruplex folding is driven almost entirely by the energy of cation binding with little or no contribution from other weak molecular interactions.