In the presence of specific metal ions, DNA oligonucleotides containing guanine repeat sequences can adopt G-quadruplex structures. In this work, we used a combination of spectroscopic and calorimetric techniques to investigate the conformation and unfolding thermodynamics of the K +-form of five G-quadruplexes with sequences: d(G 2T 2G 2TGTG 2T 2G 2), G2, d(G 3T 2G 3TGTG 3T 2G 3), G3, their analogs where T is replaced with U, G2-U and G3-U, and r(G 2U 2G 2UGUG 2U 2G 2), rG2. These G-quadruplexes show CD spectra characteristic of the “chair” conformation ( G2 and G2-U), or “basket” conformation ( rG2); or a mixture of these two conformers ( G3 and G3-U). Thermodynamic profiles show that the favorable folding of each G-quadruplex results from the typical compensation of a favorable enthalpy and unfavorable entropy contributions. G-quadruplex stability increase in the following order (in Δ G° 20): rG2 (−1.3 kcal/mol) < G2 < G2-U < G3-U (chair) < G3 (chair) < G3-U (basket) < G3 (basket) (−8.6 kcal/mol), due to favorable enthalpy contribution from the stacking of G-quartets. We used ITC to determine thermodynamic binding profiles for the interaction of the minor groove ligands, netropsin and distamycin, with each G-quadruplex. Both ligands bind with high exothermic enthalpies (∼−10.8 kcal/mol), 1:1 stoichiometries, and weak affinities (∼8 × 10 4 M −1). The similarity of the binding thermodynamic profiles, together with the absence of induced Cotton effects, indicates a surface or outside binding mode. We speculate that the top and bottom surfaces of the G-quadruplex comprise the potential MGBL binding sites, where the ligand lies on the surface forming van der Waals interactions with the guanines of the G-quartets and loop nucleotides.