Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was </=0.5, at which only 10-30% of DOTAP in the lipoplex is neutralized. Prolonged incubation time of lipoplexes before addition to cells slightly decreased the level of transfection. A major influence on the lipofection level was found when the mode of lipoplex preparation was varied. Mixing plasmid DNA and DOTAP/DOPE (1:1) LUV in two steps instead of one step resulted in a higher lipofection when at the first step the DNA/DOTAP mole ratio was 0.5 than when it was 2.0. Only static light-scattering measurement, which is related to particle size and particle size instability, revealed differences between the lipoplexes as a function of lamellarity of the vesicles (MLV or LUV), mixing order, and number of mixing steps. Other physical properties of these lipoplexes were dependent only on the DNA/DOTAP mole ratio, i.e. the extent of DOTAP neutralization (as monitored by ionization of the fluorophore 4-heptadecyl-7-hydroxycoumarin) and the extent of defects in lipid organization (as monitored by level of exposure of the fluorophore 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene to water). The secondary and tertiary structure of DNA in lipoplexes was evaluated by circular dichroism spectroscopy. The results of this study point out that the structure of lipoplexes should be physicochemically characterized at two different levels: the macro level, which relates to size and size instability, and the micro level, which relates to the properties described above which are involved in the intimate interaction between the plasmid DNA and the lipids. At the micro level, all parameters are reversible, history-independent and are determined by DNA/DOTAP mole ratio. On the other hand, the macro level (which is the most important for transfection efficiency) is history-dependent and not reversible.