We report a study of the behavior of oligodeoxyribonucleotide (ODN)/amphotericin B3-( N′-dimethylamino)propylamide (AMA) complexes, in the presence of lipid monolayers and large unilamellar vesicles. This study follows the recent discovery of the capacity of AMA, as a new cationic vector, to enhance ODN cellular uptake and efficacy. It aims at investigating the internalization mode of a nucleic acid by AMA. A first study at the air–water interface of AMA and AMA/ODN by surface pressure measurement shows that only free AMA would adsorb at the air–water interface. Second, in the presence of zwitterionic phospholipid- and sterol-containing mixture, ODN–AMA interactions in solution would be higher than lipid–AMA interactions at the interface. In monolayer or with large unilamellar vesicles, AMA monomers adsorb mainly at the phospholipid interface. These results favor a crossing mechanism through AMA channel formation, despite the size of ODN.