In the present paper interactions between enrofloxacin and phosphatidylcholine, dipalmitoylphosphatidylcholine, and mixtures of these lipids with cholesterol were studied by different techniques. Hydrophobic interactions were determined by differential scanning calorimetry, carboxyfluorescein leakage from liposomes, and compression isotherms of mixed monolayers. The penetration kinetics as well as the polarization changes of the anilinonaphthalene sulfonate probe inserted into liposomes were used to study the effect of the drug in the lipid polar head motion. The drug was unable to form monolayers and compression isotherms showed a low level of drug interaction with phospholipids. This was also confirmed by differential scanning calorimetry measurements in which no change in the main transition temperature was observed but a slight broadening of the peaks. However, a strong influence in the fluidity of the polar part of the bilayers after incubation of liposomes with enrofloxacin was found.