Cationic liposomes are potentially important gene transfer vehicles, capable of conjugating with anionic DNA by condensation. Flow cytometry was used to examine quantitatively the incorporation of DNA-liposome complex into murine capillary lung endothelial cells. The plasmid DNA, a pSV-beta-galactosidase vector, was covalently labeled with ethidium monoazide by photoactivation. The cationic liposome consisted of egg phosphatidylcholine (90%), cholesterol (5%), and stearylamine (5%). The number of plasmid molecules contained within each cell as a function of exposure time was estimated from fluorescence intensity. Fluorescently labeled plasmid is detectable after 10 min and increases with continued exposure, but at a decreasing rate, up to 2160 min. After 2160 min each cell, on average, contains approximately 10,000 plasmid molecules. Following transfection, a single cell unimodal population was detected by flow cytometry, suggesting that all cells participate in transfection equally. Furthermore, cell cycle analysis indicates that the entry of DNA-liposome complex is independent of cell cycle.