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The delivery of therapeutic molecules such as plasmid DNA in cells and tissues by means of electric fields holds great promise for anticancer treatment. To allow for their therapeutic action, the molecules have first to traverse the cell membrane. The mechanisms by which the electrotransferred pDNA interacts with and crosses the plasma membrane are not yet fully explained. The aim of this study is to unravel the role of cholesterol during gene electrotransfer in cells. We performed cholesterol depletion experiments and measured its effects on various steps of the electroporation process. The first two steps consisting of electropermeabilization of the plasma membrane and of pDNA interaction with it were not affected by cholesterol depletion. In contrast, gene expression decreased. Colocalization studies with endocytotic markers showed that pDNA is endocytosed with concomitant clathrin- and caveolin/raft-mediated endocytosis. Cholesterol might be involved in the pDNA translocation through the plasma membrane. This is the first direct experimental evidence of the occurrence of endocytosis in gene electrotransfer.