The interaction of two fluoroquinolones, ciprofloxacin (CPX) and its N-4-butylpiperazinyl derivative (BCPX), with liposomes bearing negative surface charge was studied by fluorescence methods. Binding of 1-anilino-8-naphthalenesulfonate to the liposome surface and diphenylhexatriene (DPH) and diphenylhexatriene-propionic (DPH-AP) acid fluorescence anisotropy measurements were differently affected by the two. On the basis of the variations of the surface potential and phase transition temperature (T) of the phospholipids studied, it was concluded that (i) both drugs interact electrostatically at the lipid membrane interface and (ii) the butyl side chain of BCPX led to a different interaction with the lipid headgroups. This behavior is then related to the activity of an efflux pump Serratia marcescens NIMA, which appear to be affected by the ability of CPX and BCPX to interact with the inner surface of the plasma membrane.