The interactions and complexation process of the amphiphilic penicillins sodium cloxacillin and sodium dicloxacillin with horse myoglobin in aqueous buffered Solutions of pH 4.5 and 7.4 have been examined by equilibrium dialysis, xi-potential, isothermal titration calorimetry (ITC) and UV-Vis absorbance techniques. A more opened structure of the protein molecules is detected as a consequence of the reduction of pH from 7.4 to 4.5. Binding isotherms and derived Hill coefficients reflect a cooperative binding behavior. Gibbs energies of binding per mole of drug were obtained from equilibrium dialysis data and compared with those derived from the potential taking into account cooperativity. DeltaG(ads)degrees values so obtained are large and negative at low concentrations where binding to the "high-energy" sites occurs and decreases with the drug concentration. The enthalpies of binding have been obtained from ITC and are small and exothermic so that the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Other thermodynamic quantities of the binding mechanism, that is, entropy, DeltaS(ITC)(i), Gibbs energy, DeltaG(ITC)(i), the binding constant, K-ITC(i), and the number of binding sites, n(i), were also obtained, confirming the above results. From ITC data and following a theoretical model, the number of bound and free penicillin molecules was calculated, being higher at pH 4.5 than at pH 7.4. The binding of penicillin causes a conformational transition on protein structure as a consequence of the resulting intramolecular repulsion between the penicillin molecules bound to the protein. Thermodynamic quantites (the Gibbs energy of the transition in water, DeltaG(W)degrees, and in a hydrophobic environment, DeltaG(hc)degrees) of the denaturation process were calculated, indicating that at pH 4.5 some of the histidine residues are protonated, becoming accessible to solvent and giving rise to a more opened protein structure.