To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.