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Plasmid DNA (pDNA) can occur in the compact supercoiled (SC) form, the relaxed open circular (OC) form and the linearized form. In this paper we investigated the transfection efficiency of SC, OC and linearized pDNA complexed to DOTAP/DOPE liposomes in Vero cells. Only DOTAP/DOPE liposomes containing SC pDNA showed protein expression while DOTAP/DOPE liposomes loaded with OC or linearized pDNA failed. First we questioned if the better transfection properties of the SC pDNA-containing lipoplexes could be due to a better transcription of SC pDNA in the nuclei of the cells, compared to OC and linearized pDNA. However, microinjecting (naked) SC, OC or linearized pDNA in the nuclei of the Vero cells revealed that the transcription efficiency was independent on the pDNA topology but did depend on the intranuclear concentration of the pDNA. As the amount of pDNA that reaches the nucleus is determined by the amount of pDNA that arrives in the cytosol it could be hypothesized that SC pDNA is more efficiently released from the DOTAP/DOPE liposomes when compared to OC and linearized pDNA. However, microinjecting comparable concentrations of the pDNA topologies in the cytoplasm still resulted in a significantly higher transfection in the case of SC pDNA, especially in cells that underwent cell division in the period after injection. It seems that, compared to OC and linearized pDNA, SC pDNA is better suited to reach the perinuclear region, a prerequisite to become entrapped in the nuclei of the cells during cell division.