Lipoplexes containing a mixture of cationic phospholipids dioleoylethylphosphatidylcholine (EDOPC) and dilauroylethylphosphatidylcholine (EDLPC) are known to be far more efficient agents in transfection of cultured primary endothelial cells than are lipoplexes containing either lipid alone. The large magnitude of the synergy permits comparison of the physical and physico-chemical properties of lipoplexes that have very different transfection efficiencies, but minor chemical differences. Here we report that the superior transfection efficiency of the EDLPC/EDOPC lipoplexes correlates with higher surface activity, higher affinity to interact and mix with negatively charged membrane-mimicking liposomes, and with considerably more efficient DNA release relative to the EDOPC lipoplexes. Observations on cultured cells agree with the results obtained with model systems; confocal microscopy of transfected human umbilical artery endothelial cells (HUAEC) demonstrated more extensive DNA release into the cytoplasm and nucleoplasm for the EDLPC/EDOPC lipoplexes than for EDOPC lipoplexes; electron microscopy of cells fixed and embedded directly on the culture dish revealed contact of EDLPC/EDOPC lipoplexes with various cellular membranes, including those of the endoplasmic reticulum, mitochondria and nucleus. The sequence of events outlining efficient lipofection is discussed based on the presented data.