We recently reported that a cationic liposomal vector, TFL-3, could be used to achieve significant gene expression in primary cultured rat hepatocytes (Nguyen et al., Biol. Pharm. Bull., 26, 880-885 (2003)). A combination of hepatocyte transplantation and hepatocyte-targeted gene transfer represents a potentially important strategy for expanding treatment options for liver disease. A widely applied approach to support cross-species is necessary before human applications can be realized. Therefore, in this study, we examined the utility of TFL-3 in another species of rodent hepatocytes, namely mouse hepatocytes. Gene expression in mouse hepatocytes by TFL-3 was successful and the level was higher than those in rat hepatocytes that we recently reported on. Interestingly, it appears that both the degree and rate of gene expression were dependent on the incubation time prior to lipofection as well as on the density of cells per dish, but these parameters were independent of the amount of pDNA associated with the cells. These significantly suggest that the culture time prior to and following lipofection, which are related to the biological condition of the cells, may be one of major factors that affect gene expression in hepatocytes and non- or less dividing cells.