We have previously shown that cationic cholesterol derivatives bearing guanidinium groups were efficient vectors for gene transfer. To further evaluate the potentiality of this novel class of cationic lipids, we undertook to study the transfection efficiency of guanidinium-based lipids with other hydrophobic moieties, Specifically, we synthesized a reagent where two guanidinium groups are linked to a diacetylene lipid which may provide the lipoplexes with favorable structural features. We report here that the cationic lipid disguanidinium-diacetylene (BGDA) is highly efficient for in vitro gene transfection when formulated with dioleoylphosphatidyl ethanolamine (DOPE), We also show that liposomes composed of BGDA, DOPE, and a neutral diacetylene colipid, hydroxyethylenediacetylene (HEDA), are efficient for transfection, Thus, diacetylene-based lipids provide a novel scaffold for gene transfection and will be particularly useful for gaining new insights into the structure-activity relationships of the lipid/DNA complexes as they offer a means to study the effects of polymerizable domains.