Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.