Keratinocytes have a great potential to deliver systemically therapeutic genes, and a regulatable switch technology for transgene expression in this cell type would greatly enhance their clinical value for cutaneous gene therapy. We describe a method wherein immortalized human keratinocytes (IMKc) are transduced with high efficiency with retroviral vectors of the RetroTet-Art system, which confers stable doxycycline (Dox)-regulated green fluorescent protein (GFP) expression. In this RetroTet-Art system the TCN transactivators and TCN transrepressors are coexpressed in cells. After one round of transduction, approximately 50% of IMKc expressed GFP; after puromycin selection over 90% of cells expressed GFP. With this retroviral vector system no baseline expression of GFP was observed in the genetically modified IMKcs. Dox treatment of these transduced cells induced GFP expression in a dose- and time-dependent manner. Peak GFP expression occurred after 72 h of Dox treatment and dropped to baseline when Dox was removed. These multiply transduced cells formed differentiated epidermis in vitro and the Dox treatment did not induce evidence of toxicity in the architecture of the epidermis. Our observations demonstrate an efficient method for achieving stable Dox-regulatable transgene expression in human keratinocytes.