To date, the primary approach to improving the transfection properties of cationic lipids has been the synthesis of new kinds of cationic amphipaths. Recently, however, it was found that combining two cationic lipid derivatives having the same head group but tails of different chain lengths can provide another, and often superior, approach to higher transfection efficiency. For example, the combination of medium-chain and long-chain homologues of O-ethylphosphatidylcholine transfected DNA into primary human umbilical artery endothelial cells (HUAECs) more than 30-fold more efficiently than did either compound separately. Here it is reported that this synergism of mixtures is not limited to O-ethylphosphatidylcholine homologues, but is also exhibited by other common cationic amphipathic transfection reagents; for example, combining DC-Chol (3beta-[N',N'-dimethylaminoethane)-carbamol] cholesterol), dimethylditetradecylammonium bromide, or DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane) with EDOPC increased transfection significantly both in the absence and in the presence of serum. Furthermore, combining a poorer transfection agent-dimethyldioctadecylammonium bromide-with dimethylditetradecylammonium bromide increased transfection by about an order of magnitude with a maximum at an intermediate composition. Lack of synergy occurred with some mixtures, such as DMTAP and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), in which case transfection activity was a linear function of composition both in the absence and presence of serum. Although the mechanism of enhanced transfection by mixtures is not fully understood, the existence of a number of optimal mixtures with diverse cationic compounds indicates that attention to mixture formulations can lead to greatly improved transfection by cationic amphipathic carriers.