The DNA binding of toluidine blue O (TBO), azure A and azure B was characterised by isothermal titration calorimetry, differential scanning calorimetry and thermal melting studies. The DNA binding affinity of TBO was the highest followed by azure A and azure B. The binding in each case was exothermic with a positive entropy change. The affinity of the binding decreased as the [Na+] concentration increased. The non electrostatic contribution to the standard Gibbs energy remained the same over the range of (10 to 100) mM [Na+]. The negative change in heat capacity of the binding revealed a substantial hydrophobic contribution in the DNA binding of these dyes. An enthalpy-entropy compensation was observed in each system. The binding of these dyes stabilised the DNA against thermal strand separation. The energetics of the DNA binding of these dyes correlate well with the structural data that suggest their utility as potential DNA targeting agents. (C) 2013 Elsevier Ltd. All rights reserved.