Cytosine-rich DNA at low pH adopts an antiparallel tetraplex structure via the intercalation of two partially protonated, parallel stranded duplexes. This intriguing structural motif has been named i-DNA. We have used a combination of spectroscopic and calorimetric techniques to characterize the properties of an intermolecular i-DNA formed by d(TC(5)) and an intramolecular i-DNA formed by d[(C(5)T(3))(3)C(5)]. Our measurements reveal that both i-DNA complexes are enthalpically stabilized by 6.5-7.0 kcal/mol(base) and entropically destabilized by 20 cal/mol(base)/K. These values are about 50% larger than the corresponding enthalpy and entropy values per base for Watson and Crick duplexes and for Hoogsteen triplexes, while being similar to per base enthalpy and entropy values reported for G-quadruplexes. Our data also reveal a positive heat capacity change between 20 and 30 cal/mol(base)/K, values similar to that reported for polymeric Watson & Crick DNA duplexes. Solution-dependent studies reveal the overall thermal and thermodynamic stability of i-DNA complexes to be dictated by an interplay between pH and ionic strength. Based on the thermodynamic data measured, we discuss the feasibility of i-DNA formation in the context of conventional DNA sequences, while commenting on potential roles for this structural motif in biological regulatory mechanisms.