In a reported gene assay, cationic liposomes containing the cationic lipid 3 beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) and a neural phospholipid dioleoylphosphatidylethanolamine (DOPE) showed high transfection activity. DNA/liposome complex which contained low amount of liposomes could bind to the cell surface but failed to transfect the cells. We have designed a two-step protocol to examine this phenomenon in more detail. A431 human cells were incubated on ice (pulse) with DNA complexed to a low level of cationic liposomes. The cells were washed and incubated at 37 degrees C (chase) with or without free cationic liposomes of various composition (helper liposomes). Only liposomes enriched with DOPE showed helper activity; liposomes containing dioleoylphosphatidylcholine (DOPC), a structural analog of DOPE, had no helper activity. The delivery was inhibited by the lysosomotropic agent chloroquine and was optimal if the helper liposome chase was initiated immediately after the pulse. An endocytosis model of DNA delivery by cationic liposomes is proposed in which the principal function of the chase liposomes is to destabilize the endosome membrane and allow the release of DNA into the cytosol. This model is consistent with the known activity of DOPE to assume non-bilayer structures, hence destabilizing the endosome membrane.