It has been shown that DNA oligonucleotides composed, in part, of G repeat sequences can adopt G-quadruplex structures in the presence of specific metal ions. In this work, we use a combination of spectroscopic and calorimetric techniques to determine the spectral and thermodynamic characteristics of two DNA aptamers, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), G2, and d(G(3)T(2)G(3)TGTG(3)T(2)G(3)), G3; a sequence in the promoter region of the c-MYC oncogene, d(TG(4)AG(3)TG(4)AG(3)TG(4)A(2)G(2)), NHE-III; and the human telomere sequence d(AG(3)T(2)AG(3)T(2)AG(3)T(2)AG(3)) 22GG. The circular dichroism spectra of these oligonucleotides in the presence of K+ indicate that all form G-quadruplexes with G-quartets in an antiparallel arrangement (G2), in a parallel arrangement (NHE-III and 22GG), or in a mixed parallel and antiparallel G-quartet arrangement (G3). Melting profiles show transition temperatures, T-M, above 45 degrees C that are independent of strand concentration, consistent with the formation of very stable intramolecular G-quadruplexes. We used differential scanning calorimetry to obtain complete thermodynamic profiles for the unfolding of each quadruplex. Subtracting the thermodynamic folding profiles of G2 from those of G3 yielded the following thermodynamic profile for the formation of a G-quartet stack: Delta G degrees(20) = -2.2 kcal/mol, Delta H-cal = -14.6 kcal/mol, T Delta S-cal = -12.4 kcal/mol, An(K+) = -0.3 mol of K+/mol, and Delta nw, = 13 mol of H2O/mol. Furthermore, we used this profile to estimate the thermodynamic contributions of the loops and/or extra base sequences of each oligonucleotide in the G-quadruplex state. The average free energy contributions of the latter indicate that the incorporation of loops and base overhangs stabilizes quadruplex structures. This stabilization is enthalpy-driven and is due to base-stacking contributions.