Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.