Following our previous finding that increasing charge ratio (+/-) can overcome the serum sensitivity of cationic liposome-DNA complex, in the present studies we report that serum sensitivity of cationic liposome-DNA complex could also be overcome by prolonging the incubation time of the complex. Such time-dependent maturation of the complex was cationic lipid-dependent; complexes containing monovalent cationic lipids, eg DC-chol, DOTAP and lipofectin, matured with time, but those containing multivalent lipid, eg lipofectamine, do not. High charge ratio high concentration and high temperature accelerated the process of maturation. The serum sensitivity of cellular uptake of DNA was also dependent on the incubation time. The matured cationic liposome-DNA complexes are homogeneous particles with a mean diameter of 170 to 400 nm, depending on the cationic lipid in the complex.