The interaction between ofloxacin, as a model drug of the fluoroquinolone class, and biomembranes was examined as the possible initial step in a transmembrane diffusion process. Dipalmitoylphosphatidylcholine was used for the preparation of biomembrane models. The influence of environmental conditions and protonation on molecular physicochemical behavior, and hence on the membrane interaction, was investigated by differential scanning calorimetry (DSC). This technique has been shown to be very effective in the interpretation of interactions of drug microspeciations with biomembranes. These findings suggest that the interaction occurred owing to ionic and hydrophobic forces showing how the passage through the membrane is mainly favored in the pH interval 6-7.4. It was demonstrated that a pH gradient through model membranes may be responsible for a poorly homogeneous distribution of ofloxacin (or other related fluoroquinolones), which justifies the in vivo accumulation properties of this drug. DSC experiments, which are in agreement with computational data, also showed that the complexing capability of ofloxacin with regard to Mg(++) or Ca(++) may govern the drug entrance into bacterial cells before the DNA Girase inhibition and could ensure the formation of hydrophobic and more fluid phospholipid domains on the surface of the model membrane. These regions are more permeable with regard to various solutes, as well as ofloxacin, allowing a so-called 'self-promoted entrance pathway'. The combination of experimental methodologies with computational data allowed a further rationalization of the results and opened new perspectives into the mechanism of action of ofloxacin, namely its interaction with lipid bilayers and drug-divalent cation complex formation, which might be extended to the entire fluoroquinolone class. Ofloxacin accumulation within Escherichia coli ATCC 25922 was measured as a function of time. Also in this example, the environmental conditions influenced ofloxacin penetration and accumulation. The in vitro experiments, reported here, show that a suitable balance of hydrophilic and hydrophobic fluoroquinolone properties needs to occur for there to be increased drug permeation.