Cationic lipids are widely used for gene delivery, and inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid in cationic lipid-DNA formulations often promotes transfection efficacy. To investigate the significance of DOPE's preference to adopt a hexagonal phase in the mechanism of transfection, the properties and transfection efficiencies of SAINT-2/DOPE lipoplexes were compared to those of lipoplexes containing lamellar-phase-forming dipalmitoylphosphatidylethanolamine (DPPE). After interaction with anionic vesicles, to simulate lipoplex-endosomal membrane interaction, SAINT-2/DOPE lipoplexes show a perfect hexagonal phase, whereas SAINT-2/DPPE lipoplexes form a mixed lamellar-hexagonal phase. The transition to the hexagonal phase is crucial for dissociation of DNA or oligonucleotides (ODN) from the lipoplexes. However, while the efficiencies of nucleic acid release from either complex were similar, SAINT-2/DOPE lipoplexes displayed a two- to threefold higher transfection efficiency or nuclear ODN delivery. Interestingly, rupture of endosomes following a cellular incubation with ODN-containing SAINT-2/DPPE complexes dramatically improved nuclear ODN delivery to a level that was similar to that observed for SAINT-2/DOPE complexes. Our data demonstrate that although hexagonal phase formation in lipoplexes is a prerequisite for nucleic acid release from the complex, it appears highly critical for accomplishing efficient translocation of nucleic acids across the endosomal membrane into the cytosol for transport to the nucleus.