Epithelial cell lines can be grown on filter supports and form polarized monolayers with distinct basolateral and apical plasma membrane domains. This property has been extensively used in cell biology to investigate epithelial cell function. To date, a major limitation of this approach has been the difficulty of obtaining transient gene expression in polarized epithelia. Here we present an approach to overcome this problem using gene transfer into polarized epithelial cells grown on filters using a herpes virus-based vector. Recombinant genes are inserted into a defective HSV-1 plasmid and packaged with a replication-incompetent HSV-1 helper virus into virus particles which are used to infect the polarized epithelial cells grown on filters. The transepithelial resistance of the cells is not affected by the addition of virus, and there are no detectable cytopathic effects.