A critical requirement of gene therapy is expression of the delivered transgene. Transgene expression is facilitated by access to the transcription mechanism found primarily in the nucleus. Factors modulating the interactions between intracellular plasmid and nuclear access are not well understood. In this study, the effect of mitosis on transgene expression was examined by quantitative flow cytometry. Transfection of HeLa cells synchronized at late G1 phase or G2/M phase was performed using a liposomal vector containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dioleoyl-phosphatidylethanolamine (DOPE) (1:1 mol/mol). Cell samples were transfected and subsequently maintained in G1 phase for various durations to modulate the time between plasmid entry and mitosis. The plasmid contains the sequence for a mutated green fluorescent protein (GFP(S65T)) that was used to examine transgene expression. Ethidium monoazide-labeled plasmid was employed to examine the association of plasmid with the cell membrane. The percentage of cells expressing GFP(S65T) increased sharply as the synchronized cell population passed through M phase, suggesting that an event associated with mitosis is essential for transgene expression. Expression levels of the transgene then declined 18 h after mitosis irrespective of transfection strategy. All transfection strategies resulted in the same maximum percentage of GFP(S65T) positive cells (40%) and average GFP(S65T) expression level (3.14x106 molecules per positive cell). Association of plasmid with the cell membrane at late G1 phase was 1.5-fold of that at G2/M phase. These data are evidence for control of transgene expression triggered by events associated with cell cycle.