The stability of nonviral vectors during freeze-drying has been well-studied, and it has been established that sugars can protect lipoplexes during freeze-drying. However low levels of damage are often observed after freeze-drying, and this damage is more evident in dilute lipoplex preparations. By investigating the stability of lipoplexes after each step in the freeze-drying cycle (i.e., freezing, primary drying, and secondary drying), we strive to understand the mechanisms responsible for damage and identify improved stabilization strategies. N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)-cholesterol/plasmid DNA lipoplexes were prepared at an equimolar DOTAP-cholesterol ratio, and a 3:1 DOTAP ΓΎ -DNA Γ charge ratio. Our experiments indicate that despite sufficient levels of ''stabilizing'' sugars, significant damage is still evident when dilute lipoplex preparations are subjected to freeze-drying. Analysis of the different stages of freeze-drying suggests that significant damage occurs during freezing, and that sugars have a limited capacity to protect against this freezinginduced damage. Similar effects have been observed in studies with proteins, and surfactants have been employed in protein formulations to protect against surfaceinduced damage, for example, at the ice crystal, solid, air, or sugar glass surfaces. However, the use of surfactants in a lipid-based formulation is inherently risky due to the potential for altering/solubilizing the lipid delivery vehicle. Our data indicate that judicious use of surfactants can reduce surface-induced damage and result in better preservation of lipoplex size and transfection activity after freeze-drying.