We have permeabilized and transfected mammalian cells by transient alteration of their native transmembrane electrical potential difference. Chinese hamster ovary cells were chosen as a model in order to study the electropermeabilization and electrotransfection processes. Propidium iodide was used to monitor permeabilization. A plasmid carrying the beta-galactosidase gene was used to follow direct gene transfer and expression by determining transient expression of the electrotransfered activity at the single-cell level. The effect of nucleotides on cell permeabilization and transfection was studied by altering the cytosolic ATP and ADP contents of cells either during the pulsation or during the period following it. Permeabilization and transfection are not regulated in the same way by the ATP and ADP levels. The permeabilization efficiency remains unaffected. Cell viability and the transfection yield are dramatically affected. While ADP is involved in the step of DNA transfer across the electropermeabilized plasma membrane, ATP controls other steps (cytoplasmic DNA migration towards the nucleus, expression). Our results prove, firstly, that membrane resealing is required but is not sufficient to preserve cell viability and, secondly, that transfection is a cell-mediated process and not only an electrophoretic step driven by the external field.