Two cationic phospholipid derivatives with asymmetric hydrocarbon chains were synthesized: ethyl esters of oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-EPC) and stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC). The former was 50 times more effective as a DNA transfection agent (human umbilical artery endothelial cells) than the latter, despite their similar chemical structure and virtually identical lipoplex organization. A likely reason for the superior effectiveness of C18:1/C10-EPC relative to C18:0/C10-EPC (and to many other cationic lipoids) was suggested by the phases that evolved when these lipoids were mixed with negatively charged membrane lipid formulations. The saturated C18:0/C10-EPC remained lamellar in mixtures with biomembrane-mimicking lipid formulations [e.g., dioleoyl-phosphatidylcholine/dioleoyl-phosphatidylethanolamine/dioleoyl-phosphatidylserine/cholesterol at 45:20:20:15 (wt/wt)]; in contrast, the unsaturated C18:1/C10-EPC exhibited a lamellar-nonlamellar phase transition in such mixtures, which took place at physiological temperatures, approximately 37 degrees C. As is well known, lipid vehicles exhibit maximum leakiness and contents release in the vicinity of phase transitions, especially those involving nonlamellar phase formation. Moreover, nonlamellar phase-forming compositions are frequently highly fusogenic. Indeed, FRET experiments showed that C18:1/C10-EPC exhibits lipid mixing with negatively charged membranes that is several times more extensive than that of C18:0/C10-EPC. Thus, C18:1/C10-EPC lipoplexes are likely to easily fuse with membranes, and, as a result of lipid mixing, the resultant aggregates should exhibit extensive phase coexistence and heterogeneity, thereby facilitating DNA release and leading to superior transfection efficiency. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for transfection success and suggest a strategy for the rational design of superior cationic lipid carriers.