We studied transplantation of retrovirus vector transduced canine keratinocytes to determine whether keratinocytes could persist and express the transferred gene after superficial transplantation to full-thickness wounds of dogs, a large random-bred model for gene transfer studies. Canine keratinocytes were transduced by co-cultivation with PA317 retrovirus packaging cells which produced helper-free amphotropic retroviral vectors (LZSN and LNPOZ) encoding the genes for beta-galactosidase (beta-gal) and neomycin phosphotransferase (neo). Efficient transfer and expression of the two genes could be demonstrated in confluent keratinocyte cultures for both vectors. When transduced keratinocytes were grown in organotypic cultures on a collagen matrix containing autologous dermal fibroblasts at the air-liquid interface, the cultures showed well-organized and defined epidermal cell layers and several markers of terminal differentiation, including the presence of keratohyalin granules and a multilayered stratum corneum. To determine whether the transferred beta-gal gene was also expressed in vivo, we performed autologous transplantation of transduced keratinocytes onto full-thickness wounds of dogs. beta-Gal expressing keratinocytes could be demonstrated in situ in the regenerating epidermis 2 weeks after transplantation. We conclude that keratinocytes can be efficiently transduced by retroviral vectors, that retroviral transduction does not interfere with proliferation or differentiation, and that transduced keratinocytes express the transferred gene after transplantation to full-thickness skin wounds of dogs. Keratinocytes thus seem to be good target cells for gene therapy.