The purpose of this study is to investigate the effects of moisture content on the storage stability of freeze-dried lipoplex formulations. DC-Cholesterol: DOPE (dioleoyl phosphatidylethanolamine)/plasmid DNA lipoplexes were prepared at a 3-to-2 DC-Cholesterol(+) to DNA(-) molar ratio and lyophilized prior to storing at room temperature, 40, and 60 degrees C for 3 months. Different residual moistures (1.93%, 1.10%, 1.06%, and 0.36%) were obtained by altering the secondary drying temperatures. In addition to moisture content, lipoplex formulations were evaluated after freeze-drying and/ or storage for particle size, transfection efficiency, accumulation of thiobarbituric acid reactive substances (TBARS), glass transition temperature, DNA supercoil content, and surface area. Lipoplex formulations stored at room temperature for 3 months maintain TBARS concentrations and supercoil contents. At higher storage temperatures, formulations possessing the highest moisture content (1.93%) maintained significantly lower TBARS concentrations and higher supercoil content than those with the lowest (0.36%) moisture content. Curiously, the intermediate moisture contents exhibited marked differences in stability despite virtually identical moisture contents. Subsequent measurements of surface area indicated that the lower stability corresponded to higher surface area in the dried cake, suggesting that there may be an interplay between water content and surface area that contributes to storage stability.