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The interaction of calf thymus deoxyribonucleic acid (ct-DNA) with betaxolol (BET) in aqueous buffer solution (pH 7.40) has been investigated using isothermal titration calorimetry (ITC), ultraviolet absorption (UV), fluorescence spectroscopy (FS) and circular dichroism (CD). Thermodynamic parameters, i.e., equilibrium constants, standard changes of enthalpy (ΔH°), Gibbs free energy (ΔG°) and entropy (ΔS°), for the binding process of the drug to the bio-macromolecules have been derived from the calorimetric data. Analysis of the thermodynamic data indicates that there are two classes of binding sites on the DNA molecules being able to coordinate with BET molecules. One class of binding takes place at the sites formed by base pairs, which is synergistically driven by enthalpy and entropy, while the other one takes place on phosphate groups and shown as an entropy driven process. The thermodynamic behavior of the DNA–drug supramolecular system has been discussed in the light of the important weak interactions, hydrophobic force, hydrogen bond and electrostatic force, according to the UV, FS and CD spectra.