We report herein the invention of and proof of function for a porous membrane-based electroporation device that can deliver molecules into spatially restricted and predefined areas of a cell monolayer. The device's cell culture substrate is a microporous alumina membrane (pore size 0.02 mu m), with an underlying thin poly-(dimethylsiloxane) (PDMS) film that has one or more holes with diameters in the one-tenth millimeter range. When a transient electric field is generated between the device's two planar electrodes - one of which is placed above the cells and the other below the PDMS layer - the filed condenses only in the volume defined by hole in the PDMS film and therefore localized electroporation can occur. We demonstrate that Lucifer Yellow (LY) and plasmid DNA are selectively introduced into only those HeLa cells located above the holes. Using the device containing a PDMS film with multiple holes, a patterned array of LY-stained cells was resulted. Compared with the operation of and the results obtained from a conventional cuvette electroporation device, our device greatly decreases the necessary operating voltage, can be used with cells attached to a substrate, and increases the number of conditions that can be screened in a single experiment. Finally, since a PDMS film with different sized holes, produces different localized electric field strengths, it is possible to determine the optimum electroporetic conditions in a single experiment. (c) 2007 Elsevier B.V. All rights reserved.