Our research on topical application of lidocaine-loaded non-ionic surfactant vesicles (NSVs) was prompted by the great interest on new delivery systems for local anaesthetics. This study is focused on a novel formulation of NSVs entrapping lidocaine in the form of a free base (LID) and a hydrochloride (LIDHCl). NSVs were prepared from polyoxyethylene sorbitan monolaurate (Tween20) and cholesterol. The effect of vesicle composition and environmental pH condition (8.6-5.5) on drug encapsulation efficiency (e.e.) was investigated. Experimental strategies involved: freeze-fracture, microscopy technique, dynamic light scattering, permeation through Silastic and mouse abdominal skin, in vitro release kinetics of vesicle-entrapped drugs, fluorescence quenching analyses. Diffusion experiments showed that the flux of charged lidocaine through Silastic membrane was possible only after the vesicle encapsulation. Permeation through mouse abdominal skin of LIDHCl loaded vesicles showed a higher flux and a shorter lag time with respect to classical liposome formulations, while LID permeation rate was quite similar for NSV and liposome formulations. Vesicles were also prepared in the presence of dicetylphosphate (DCP) and N-cetylpyridinium chloride (CP) to obtain negatively and positively charged vesicles respectively, but in this case the e.e. of the drug was negligible. The possible reason of the remarkable lower e.e. observed with charged vesicles was investigated by means of fluorescence quenching experiments.