We discovered that several high molecular weight cationic polymers, such as poly(L-lysine) and protamine, can enhance the transfection efficiency of several types of cationic liposomes by 2-28-fold in a number of cell lines in vitro. Small polycations such as spermine and a cationic decapeptide derived from SV40 T-antigen were only moderately active. The addition of poly(L-lysine) and protamine dramatically reduced the particle size of the complex formed between DNA and cationic liposomes and rendered DNA resistant to the nuclease activity. The complexes composed of DNA, poly(L-lysine), and cationic lipids were purified from an excess of free liposomes with sucrose gradient ultracentrifugation. Purified complex formed at low cationic liposome ratio was poor in lipid content and only had weak transfection activity. Addition of free liposome to the purified complex significantly enhanced the transfection activity. In contrast, complexes formed at a higher initial ratio of liposome to DNA had a higher lipid content and were highly active in transfection; the activity was about 3-9-fold more active than the corresponding complex before purification. Negative stain EM studies revealed that the most active complexes prepared from 40 nmol of lipid, 0.5 micrograms of poly(L-lysine), and 1 microgram of DNA and purified by gradient ultracentrifugation were spherical, electron dense, small (< 100 nm in diameter) particles, and some of them were associated with lipid membranes. These highly active, stable, small-sized lipid/poly(L-lysine)/DNA complexes represent a new class of nonviral gene delivery vehicles that might be useful in gene therapy.