The epidermis is an attractive target for gene therapy because it is easily accessible and shows great potential as an ectopic site for protein delivery in vivo. Genetically modified epidermal keratinocytes are used to prepare three-dimensional skin equivalents, which when transplanted into animals act as in vivo "bioreactors" that produce and deliver the desired therapeutic proteins either systemically or locally. Although retroviral transduction results in permanent genetic modification, differentiation and loss of transduced cells from the epidermis results in temporary transgene expression. To ensure permanent genetic modification epidermal stem cells must be transduced with high efficiency. To this end, we used recombinant fibronectin (FN) to immobilize retroviral particles. Our results show that cells transduced on FN exhibit significantly higher transduction efficiency, which correlates with the levels of expression of integrin subunits β1 and α5. These integrins have been previously shown to correlate with stem cell phenotype. Notably, cells that adhere rapidly to FN are transduced more efficiently than slowly adherent cells. Most importantly, the efficiency of gene transfer increases with cell passage. An increase in the fraction of transduced cells over time suggests selective transduction of progenitor cells, as the nontransduced differentiated cells exhaust their proliferative potential and stop propagating. These results may have important implications for gene therapy of the skin.