Most enzymes that participate in the biochemistry of nucleic acids require divalent metal ion cofactors to promote activity. Development of potent inhibitors, acting against those viral enzymes operating via a cooperative two-metal ion mechanism, such as HIV integrase (IN) and RNase H, hepatitis C virus polymerase and influenza endonuclease, requires optimizing the binding affinity to the target, which is dictated by the binding free energy composed of both enthalpic and entropic contributions. They can be obtained by using isothermal titration microcalorimetry. We have defined an experimental procedure for obtaining reliable thermodynamic data in methanol/water=9/1 0.1 M KCl as solvent, used to overcome solubility problems. In this way we have measured the heats of formation of the complexes formed by N-(4-fluorobenzyl)-5-hydroxy-2-isopropyl-1-methyl-6-oxo-1,6-dihydroxypyrimidine-4-carboxylate (HL, a model of Raltegravir, the antiretroviral drug produced by Merck & Co.), and a series of divalent metal ions of biological interest (Mg(II), Mn(II), Co(II) and Zn(II)), whose speciation was previously determined by potentiometry. (C) 2014 Published by Elsevier B.V.