Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing.