Cationic liposomes are potentially important gene transfer vehicles, although their application has been limited by relatively low efficiency of transgene expression. Single cell quantitative methods, such as those used in this study, should permit a more detailed understanding of the relationships between delivered plasmid and transgene expression. Intracellular plasmid delivery and transgene expression were measured simultaneously using photoconjugated ethidium monoazide as an intracellular plasmid delivery marker and green fluorescent protein (GFP(S65T)) as a transgene expression marker. Quantitative flow cytometry was used to estimate plasmid copy number and GFP(S65T) molecules in single cells. The plasmid was delivered to HeLa cells with a cationic liposome vehicle containing 1,2-dioleoyloxy-3-trimethylammonium-propane and dioleoylphosphatidylethanolamine (1:1 mol/mol). Treatment was carried out continuously for 24 h. Flow cytometry measurements on 20, 000 cells were performed during treatment and for 48 h post-treatment. On a single cell basis, transgene expression efficiency and average GFP(S65T) expression level increased with intracellular plasmid copy number. After 3-h exposure to the liposomal vector, more than 95% of the cells were positive for plasmid entry, but none had detectable transgene expression. Maximum transgene expression was achieved at 24 h and remained unchanged at the 72-h measurement. At 24 h, the average positive cell contained 1.6 x 10(5) plasmid copies and 2.3 x 10(6) GFP(S65T) molecules. Importantly, the measurement strategies revealed that transgene expression varied widely within the entire cell population. Although only 30% of all cells expressed transgene, the subpopulation of cells that rapidly incorporated the vector demonstrated 100% efficiency in transgene expression. This study identifies parameters that modulate highly efficient transgene expression from plasmid delivery by cationic liposomes.